Adipate (ester or thioester) synthesis

ABSTRACT

The present invention relates to a method for preparing an adipate ester or thioester. The invention further relates to a method for preparing adipic acid from said ester or thioester. Further the invention provides a number of methods for preparing an intermediate for said ester or thioester. Further the invention relates to a method for preparing 6-amino caproic acid (6-ACA), a method for preparing 5-formyl valeric acid (5-FVA), and a method for preparing caprolactam. Further, the invention relates to a host cell for use in a method according to the invention.

The present invention relates to a method for preparing an adipate esteror thioester. The invention further relates to a method for preparingadipic acid from said ester or thioester. Further the invention providesa number of methods for preparing an intermediate for said ester orthioester. Further the invention relates to a method for preparing6-amino caproic acid (6-ACA), a method for preparing 5-formyl valericacid (5-FVA), and a method for preparing caprolactam. Further, theinvention relates to a host cell for use in a method according to theinvention.

Adipic acid (hexanedioic acid) is inter alia used for the production ofpolyamide. Further, esters of adipic acid may be used in plasticisers,lubricants, solvent and in a variety of polyurethane resins. Other usesof adipic acid are as food acidulans, applications in adhesives,insecticides, tanning and dyeing. Known preparation methods include theoxidation of cyclohexanol or cyclohexanone or a mixture thereof (KA oil)with nitric acid.

Caprolactam is a lactam which may also be used for the production ofpolyamide, for instance nylon-6 or caprolactam-laurolactam copolymers(nylon-6,12). Various manners of preparing caprolactam from bulkchemicals are known in the art and include the preparation ofcaprolactam from cyclohexanone, toluene, phenol, cyclohexanol, benzeneor cyclohexane.

The intermediate compounds, such as cyclohexanol, cyclohexanone orphenol, for preparing adipic acid or caprolactam are generally obtainedfrom mineral oil. In view of a growing desire to prepare materials usingmore sustainable technology it would be desirable to provide a methodwherein adipic acid or caprolactam is prepared from an intermediatecompound that can be obtained from a biologically renewable source or atleast from an intermediate compound that is converted into adipic acidor caprolactam using a biochemical method.

In U.S. Pat. No. 5,487,987, a method is described for the production ofadipic acid, wherein use is made of a bacterial cell, wherein a carbonsource is converted into 3-dehydroshikimate by the enzymes in the commonpathway or aromatic amino acid biosynthesis of the bacterial cell, toproduce cis, cis muconic acid, by the biocatalytic conversion of3-dehydroshikimate. The cis, cis muconic acid is thereafter chemicallyreduced (using a platinum catalyst) to produce adipic acid. Thus, thefinal step requires chemical catalysis. It is further envisaged by thepresent inventors that the aromatic intermediates formed in thebacterial cell, may be toxic to the cell, likely requiring theirconcentration to be low in vivo as well as in the cell culture.

It is known to prepare caprolactam from 6-aminocaproic acid (6-ACA),e.g. as described in U.S. Pat. No. 6,194,572. As disclosed in WO2005/068643, 6-ACA may be prepared biochemically by converting6-aminohex-2-enoic acid (6-AHEA) in the presence of an enzyme havingα,β-enoate reductase activity. The 6-AHEA may be prepared from lysine,e.g. biochemically or by pure chemical synthesis. Although 6-ACA can beprepared via the reduction of 6-AHEA by the methods disclosed in WO2005/068643, the inventors have found that—under the reduction reactionconditions—6-AHEA may spontaneously and substantially irreversiblecyclise to form an undesired side-product, notably β-homoproline. Thiscyclisation may be a bottleneck in the production of 6-ACA, and lead toa considerable loss in yield.

It is an object of the invention to provide a novel method for preparingadipic acid or caprolactam—which may, inter alia, be used for thepreparation of polyamide—or an intermediate compound for adipic acid orcaprolactam, that can serve as an alternative to known methods.

It is a further object to provide a novel method that would overcome oneor more of the drawbacks mentioned above.

One or more further objects which may be solved in accordance with theinvention will follow from the description, below.

The inventors have realised that adipate (or a ester or thioesterthereof) can be produced from succinate (or a ester or thioesterthereof). In particular, the inventors concluded that an adipate(thio)ester may be prepared from succinate (thio)ester and acetate(thio)ester via a sequence of specific reactions, e.g. similar toreverse beta-oxidation and fatty acid biosynthesis in living cells, asshown in FIG. 2. Herein, each R independently represents an activatinggroup (facilitating the reaction), e.g. as described herein below. EachX independently represents an S or an O. ED/EDH₂ exemplifyoxidised/reduced electron donors, for example NAD/NADH, NADP/NADPH,FAD/FADH₂, or oxidised ferredoxin/reduced ferredoxin. Actual transfer ofelectrons may occur directly or may be mediated by intermediate electroncarriers such as coenzymes or electron transfer flavo proteins (ETF).Y—NH₂ refers to an amino donor, e.g. as described herein below.

Thus, the inventors came to the conclusion that it should be possible tobiocatalytically prepare adipic acid (or a ester or thioester thereof)via a cascade of reactions from succinate (or a ester or thioesterthereof) and acetate (or a ester or thioester thereof). Further, theyrealised that adipic acid may be converted biocatalytically into5-formylpentanoic acid (‘5-FVA’, 5-formylvaleric acid), which is anintermediate for the preparation of 6-ACA, and that for this conversiona specific biocatalyst may be used.

Accordingly, the present invention relates to a method for preparing anadipate ester or thioester from a succinate ester or thioester, via aplurality of reactions, wherein at least one of the reactions iscatalysed by a biocatalyst.

In particular, the invention relates to a method for preparing anadipate ester or adipate thioester, comprising converting a2,3-dehydroadipate (IUPAC name: 5-carboxy-2-pentanoate) ester or2,3-dehydroadipate thioester into the adipate ester or thioester in thepresence of a biocatalyst.

When referred herein to carboxylic acids or carboxylates, e.g. 6-ACA,another amino acid, 5-FVA, adipic acid/adipate, succinic acid/succinate,acetic acid/acetate, these terms are meant to include the protonatedcarboxylic acid (free acid), the corresponding carboxylate (itsconjugated base) as well as a salt thereof, unless specified otherwise.When referring herein to amino acids, e.g. 6-ACA, this term is meant toinclude amino acids in their zwitterionic form (in which the amino groupis in the protonated and the carboxylate group is in the deprotonatedform), the amino acid in which the amino group is protonated and thecarboxylic group is in its neutral form, and the amino acid in which theamino group is in its neutral form and the carboxylate group is in thedeprotonated form, as well as salts thereof.

When referred to ester or thioester of a carboxylic acid, e.g. adipateester or thioester, acetate ester of thioester, succinate ester orthioester, these terms are meant to include any activating group, inparticular any biological activating group, including coenzyme A (alsoreferred to as CoA), phospho-pantetheine, which may be bound to an acylor peptidyl carrier protein (ACP or PCP, respectively),N-acetyl-cysteamine, methyl-thio-glycolate, methyl-mercapto-propionate,ethyl-mercapto-propionate, methyl-mercapto-butyrate,methyl-mercapto-butyrate, mercaptopropionate and other esters orthioesters providing the same or a similar function. In case livingcells are used as a biocatalyst, the ester or thioester, in particularCoA, may be produced by the used biocatalyst or originate from anorganism also capable of producing a suitable enzyme for catalysing thereaction. CoA-ligase and CoA-transferases have been identified in manyorganisms and may provide the desired activated esters or thioesters.

The preparation of the adipate ester or thioester from the succinateester or thioester may in particular comprise the following reactionsteps (numbers between parentheses also correspond to FIG. 1):

(1) providing a succinate ester or thioester and reacting said ester orthioester with an acetate ester or thioester, thereby forming a3-oxoadipate ester or thioester;(2) hydrogenating the 3-oxo group of the 3-oxoadipate ester or thioesterthereby forming a 3-hydroxyadipate ester or thioester;(3) dehydrating the 3-hydroxyadipate ester or thioester thereby forminga 2,3-dehydroadipate ester or thioester; and(4) hydrogenating of the C—C double bond of the 2,3-dehydroadipate esteror thioester, thereby forming an adipate ester or thioester.

The invention also relates to a method for preparing an intermediatecompound, suitable for use in a method for preparing adipic acid,comprising carrying out one or more of said reactions steps 1-4, in thepresence of a biocatalyst catalyzing such reaction step.

In an embodiment, the adipate ester or thioester is converted into 5-FVA(5).

If desired, the adipate ester or thioester can be converted into adipicacid. This may be accomplished by hydrolysing the ester bond orthioester bond (7), thereby forming adipic acid or by a transferreaction, wherein ‘the alcohol’ or ‘thiol’ moiety (such as CoA) istransferred from the adipate ester or thioester to an acid differentfrom adipic acid, thereby forming adipic acid and a (thio)ester of theacid different from adipic acid (7). If succinic acid or acetate is usedas the different acid, this reaction may be advantageous in that thealcohol or thiol moiety, such as CoA may be recycled. E.g.adipyl-CoA+succinate or acetate may be converted (usually in thepresence of a CoA transferase) to form succinyl-CoA or acetyl-CoA+adipicacid. The succinyl-CoA or acetyl-CoA may then be used as a startingcompound in a method of the invention.

Adipic acid (or a ester or thioester thereof) may, e.g., be convertedinto 5-FVA (8).

In an embodiment, 5-FVA, obtained in a method of the invention isconverted into 6-ACA (6). Thereafter, 6-ACA may be converted intocaprolactam, e.g. in a manner known in the art per se.

In a further embodiment, adipic acid or caprolactam obtained in a methodaccording to the invention is used for the preparation of polyamide.

The term “a” or “an” as used herein is defined as “at least one” unlessspecified otherwise.

When referring to a noun (e.g. a compound, an additive etc.) in thesingular, the plural is meant to be included.

When referring to a compound of which several isomers exist (e.g. a cisand a trans isomer, an R and an S enantiomer), the compound in principleincludes all enantiomers, diastereomers and cis/trans isomers of thatcompound that may be used in the particular method of the invention.

When an enzyme is mentioned with reference to an enzyme class (EC)between brackets, the enzyme class is a class wherein the enzyme isclassified or may be classified, on the basis of the Enzyme Nomenclatureprovided by the Nomenclature Committee of the International Union ofBiochemistry and Molecular Biology (NC-IUBMB), which nomenclature may befound at http://www.chem.qmul.ac.uk/iubmb/enzyme/. Other suitableenzymes that have not (yet) been classified in a specified class but maybe classified as such, are meant to be included.

When referred herein to a protein by reference to a accession number,this number in particular is used to refer to a protein having asequence as found in Uniprot on 11 Mar. 2008, unless specifiedotherwise.

The term “homologue” is used herein in particular for polynucleotides orpolypeptides having a sequence identity of at least 30%, preferably atleast 40%, more preferably at least 60%, more preferably at least 65%,more preferably at least 70%, more preferably at least 75%, morepreferably at least 80%, in particular at least 85%, more in particularat least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%. Theterm homologue is also meant to include nucleic acid sequences(polynucleotide sequences) which differ from another nucleic acidsequence due to the degeneracy of the genetic code and encode the samepolypeptide sequence.

Sequence identity or similarity is herein defined as a relationshipbetween two or more polypeptide sequences or two or more nucleic acidsequences, as determined by comparing the sequences. Usually, sequenceidentities or similarities are compared over the whole length of thesequences, but may however also be compared only for a part of thesequences aligning with each other. In the art, “identity” or“similarity” also means the degree of sequence relatedness betweenpolypeptide sequences or nucleic acid sequences, as the case may be, asdetermined by the match between such sequences. Preferred methods todetermine identity or similarity are designed to give the largest matchbetween the sequences tested. In context of this invention a preferredcomputer program method to determine identity and similarity between twosequences includes BLASTP and BLASTN (Altschul, S. F. et al., J. Mol.Biol. 1990, 215, 403-410, publicly available from NCBI and other sources(BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894).Preferred parameters for polypeptide sequence comparison using BLASTPare gap open 10.0, gap extend 0.5, Blosum 62 matrix. Preferredparameters for nucleic acid sequence comparison using BLASTN are gapopen 10.0, gap extend 0.5, DNA full matrix (DNA identity matrix).

In a method of the invention, a biocatalyst is used, i.e. at least onereaction step in the method is catalysed by a biological material ormoiety derived from a biological source, for instance an organism or abiomolecule derived there from. The biocatalyst may in particularcomprise one or more enzymes. The biocatalyst may be used in any form.In an embodiment, one or more enzymes are used isolated from the naturalenvironment (isolated from the organism it has been produced in), forinstance as a solution, an emulsion, a dispersion, (a suspension of)freeze-dried cells, a lysate, or immobilised on a support. The use of anenzyme isolated from the organism it originates from may in particularbe useful in view of an increased flexibility in adjusting the reactionconditions such that the reaction equilibrium is shifted to the desiredside.

In an embodiment, one or more enzymes form part of a living organism(such as living whole cells). The enzymes may perform a catalyticfunction inside the cell. It is also possible that the enzyme may besecreted into a medium, wherein the cells are present.

Living cells may be growing cells, resting or dormant cells (e.g.spores) or cells in a stationary phase. It is also possible to use anenzyme forming part of a permeabilised cell (i.e. made permeable to asubstrate for the enzyme or a precursor for a substrate for the enzymeor enzymes).

A biocatalyst used in a method of the invention may in principle be anyorganism, or be obtained or derived from any organism. The organism maybe an eukaryote, a bacterium or an archea. In particular the organismmay be selected from animals (including humans), plants, bacteria,archaea, yeasts and fungi.

Suitable bacteria may in particular be selected amongst the group ofAbsidia, Achromobacter, Acinetobacter, Agrobacterium, Aeromonas,Alcaligenes, Arthrobacter, Arzoarcus, Azomonas, Azospirillum,Azotobacter, Bacillus, Beijerinckia, Bradyrhizobium, Burkholderia,Byssochlamys, Citrobacter, Clostridium, Comamonas, Corynebacterium,Deinococcus, Escherichia, Enterobacter, Flavobacterium, Fusobacterium,Gossypium, Klebsiella, Lactobacillus, Listeria, Megasphaera,Micrococcus, Mycobacterium, Norcadia, Porphyromonas, propionibacterium,Pseudomonas, Ralstonia, Rhizobium, Rhodopseudomonas, Rhodospirillum,Rodococcus, Roseburia, Shewanella, Streptomycetes, Xanthomonas, Xylella,Yersinia, Treponema, Vibrio, Streptococcus, Lactococcus, Zymomonas,Staphylococcus, Salmonella, Brucella, Microscilla.

Suitable eukaryotes can be selected in particular from the group offungi; metazoan; Viridiplantae (in particular Arabidopsis andChlamydomonadales); Diplomonads (in particular Giardiinae); Entamoebidae(in particular Entaboeba); Euglenozoa (in particular Euglena);Pelobiontida (in particular Mastigamoeba); and Alveolata (in particularCryptosporidium).

Suitable fungi in particular include fungi and yeasts selected amongstthe group of Rhizopus, Neurospora, Penicillium, Aspergillus, Piromyces,Trichosporon, Candida, Hansenula, Kluyveromyces, Saccharomyces,Rhodotorula, Schizosaccharomyces, Yarrowia (such as Yarrowialypolytica).

Suitable metazoan in particular include metazoan selected amongst thegroup of mammals (including human), more in particular selected from thegroup of Leporidae, Muridae, Suidae, Bovidae, hominidae. A biocatalystcan originate from any part of a metazoan, e.g. liver, pancreas, brain,kidney, heart or other organ. Suitable metazoan may also include inparticular Caenorhabditis and Drosophila.

Organisms which in particular may provide a suitable biocatalyst for aspecific reaction step are mentioned below, when describing specificreaction steps of a method of the invention.

It will be clear to the person skilled in the art that use can be madeof a naturally occurring biocatalyst (wild type) or a mutant of anaturally occurring biocatalyst with suitable activity in a methodaccording to the invention. Properties of a naturally occurringbiocatalyst may be improved by biological techniques known to theskilled person in the art, such as e.g. molecular evolution or rationaldesign. Mutants of wild-type biocatalysts can for example be made bymodifying the encoding DNA of an organism capable of acting as abiocatalyst or capable of producing a biocatalytic moiety (such as anenzyme) using mutagenesis techniques known to the person skilled in theart (random mutagenesis, site-directed mutagenesis, directed evolution,gene recombination, etc.). In particular the DNA may be modified suchthat it encodes an enzyme that differs by at least one amino acid fromthe wild-type enzyme, so that it encodes an enzyme that comprises one ormore amino acid substitutions, deletions and/or insertions compared tothe wild-type, or such that the mutants combine sequences of two or moreparent enzymes or by effecting the expression of the thus modified DNAin a suitable (host) cell. The latter may be achieved by methods knownto the skilled person in the art such as codon optimisation or codonpair optimisation, e.g. based on a method as described in WO2008/000632.

A mutant biocatalyst may have improved properties, for instance withrespect to one or more of the following aspects: selectivity towards thesubstrate, activity, stability, solvent tolerance, pH profile,temperature profile, substrate profile, susceptibility to inhibition,cofactor utilisation and substrate-affinity. Mutants with improvedproperties can be identified by applying e.g. suitable high through-putscreening or selection methods based on such methods known to theskilled person in the art.

The substrate specificity of enzymes acting on alkyl or alkyl esters orthioesters can be modified. Molecular evolution to create diversityfollowed by screening for desired mutants and/or rational engineering ofsubstrate binding pockets may be utilised. Techniques to modify thesubstrate specificity of an enzyme used in a method of the invention maybe based on those described in the art. For instance, rationalengineering employing structural and sequence information to designspecific mutations has been utilised to modify the substrate specificityof the acyl transferase domain 4 from the erythromycin polyketidesynthase to accept alternative acyl donors. It has been shown thatmodifying the proposed substrate binding site resulted in a modifiedbinding pocket able to accommodate alternative substrates resulting in adifferent product ratio (Reeves, C. D.; Murli, S.; Ashley, G. W.;Piagentini, M.; Hutchinson, C. R.; McDaniel, R. Biochemistry 2001,40(51), 15464-15470). Both rational design and molecular evolutionapproaches have been used to alter the substrate specificity of thebiocatalyst BM3 resulting in a large number of mutants capable ofoxidizing a large variety of different alkenes, cycloalkenes, arenes andheteroarenes instead or in addition to the natural substrate of mediumchain fatty acids (e.g. myristic acid) (Peters, M. W.; Meinhold, P.;Glieder, A.; Arnold, F. H. Journal of the American Chemical Society2003, 125(44), 13442-13450; Appel, D.; Lutz-Wahl, S.; Fischer, P.;Schwaneberg, U.; Schmid, R. D. Journal of Biotechnology 2001, 88(2),167-171 and references therein).

When referred to a biocatalyst, in particular an enzyme, from aparticular source, recombinant biocatalysts, in particular enzymes,originating from a first organism, but actually produced in a(genetically modified) second organism, are specifically meant to beincluded as biocatalysts, in particular enzymes, from that firstorganism.

The Preparation of 3-Oxoadipate (Ester or Thioester) Reaction 1

In an embodiment of the invention, 3-oxoadipate (ester or thioester) isprepared from succinate and acetate, which succinate and/or acetatewhich are usually provided with an activating group, in particular toyield an ester or a thioester, facilitating the reaction.

The 3-oxoadipate (ester of thioester) may be accomplishedbiocatalytically or chemically, in particular by a ‘Claisencondensation’, wherein an acetate ester or thioester and a succinateester or thioester are coupled

In a preferred method of the invention, the preparation comprises abiocatalytic reaction in the presence of a biocatalyst capable ofcatalysing an acyl-group transfer. An enzyme having such catalyticactivity may therefore be referred to as an acyltransferase.

In a preferred method an acyl transfer takes place between two acylthioesters or acyl esters. Preferred acyl thioesters are acetyl-CoA andsuccinyl-CoA. Preferably, the said biocatalyst is selective towards thesaid acyl thioesters.

The biocatalyst may in particular comprise an enzyme capable ofacyl-group transfer selected from the group of acyltransferases (E.C.2.3.1), preferably from the group of acetyl-CoA:acetyl-CoAC-acetyltransferases (EC 2.3.1.9), acyl-CoA:acetyl-CoAC-acyltransferases (EC 2.3.1.16) and succinyl-CoA:acetyl-CoAC-succinyltransferases (EC 2.3.1.174, also known as beta-ketoadipyl-CoAthiolases). Acyltransferase activity has for instance been described inbeta-oxidation, fatty acid biosynthesis, polyketide biosynhesis, orbutanoate metabolism in the KEGG (Kyoto Encyclopedia of Genes andGenomes) database.

An acyltransferase may in particular be an acyltransferase of anorganism selected from the group of archae, bacteria and eukaryota.

In particular, the enzyme may originate from a microorganism that isable to degrade organic compounds comprising an aromatic or alicyclicring structure, in particular a 5, 6 or 7 membered ring structure. Theorganic compound may optionally comprise one or more heteroatoms in thering or as a substituent or a part of a substituent. For instance, theorganic moiety may be an aromatic compound, in particular an aromaticcomprising a six-membered ring. In particular the aromatic compound maybe selected from the group of phenylacetate, benzoate, catechol,protocatechuate and gentisate. The organic compound may be a alicycliccompound, in particular a cyclic alcohol, such as cyclohexanol, a cyclicketone, such as cyclohexanone, or a cycloalkane, such as cyclohexane.The organic compound may be a lactam, such as caprolactam. In anembodiment the enzyme originates from an organism capable of degrading adicarboxylic acid (usually C₆-C₁₀), in particular a straight-chainsaturated dicarboxylic acid, such as adipic acid.

In a further embodiment, the enzyme originates from an organism capableof synthesizing 3-keto-adipate e.g. as part of a secondary metabolite(e.g. malonomycin) are preferred, for instance, from Streptomycetes (inparticular Streptomyces rimosus), from Actinomycres, from otherActinobacteria or other known secondary metabolite producers.

Preferred microorganism for providing a biocatalyst capable ofcatalysing the preparation of 3-oxoadipate (ester or thioester) furtherinclude Acinetobacter (in particular Acinetobacter sp. Strain ADP1 andA. calcoaceticus), Agrobacterium (in particular A. tumefaciens),Alicaligenes (in particular Alicaligenes strains D2 and A. eutrophus),Arthrobacter, Arzoarcus (in particular A. evansii), Azomonas,Azotobacter, Bacillus (in particular B. halodurans), Beijerinckia,Bradyrhizobium, Burkholderia, Clostridia (in particular C. kluyveri, C.acetobutylicum, C. beijerinckii), Comamonas, Corynebacterium (inparticular C. glutamicum and C. aurantiacum), E. coli, Enterobacter,Flavobacterium, Megasphera (in particular M. elsdenii), Norcadia,Pseudomonas (in particular P. putida, P. aeruginosa and Pseudomonas sp.strain B13), Ralstonia (in particular R. eutropha), Rhizobium,Rhodopseudomonas (in particular R. palustris), Rodococcus (in particularR. erythropolis, R. opacus, and Rodococcus sp strain RHA1), Aspergillus(in particular A. niger), Euglenozoa (in particular Euglena gracilis),Neurospora (in particular N. crassa), Penicillium (in particular P.chrysogenum), Rhodotorula, Saccharomyces, Trichosporon (in particular T.cutaneum).

In a specific embodiment, the biocatalyst comprises an enzyme comprisingan amino acid sequence as identified in any of the SEQUENCE ID's 1-13 ora homologue thereof.

The Preparation of 3-Hydroxyadipate (Ester or Thioester) Reaction 2

In an embodiment, 3-hydroxyadipate (ester or thioester) is prepared from3-oxoadipate (ester or thioester). Usually, the 3-oxoadipate is providedwith an activating group, as indicated above.

In principle, the 3-hydroxyadipate (ester or thioester) may be preparedchemically, e.g. by selective hydrogenation of the 3-oxo group in3-oxo-adipate (ester or thioester).

This reaction may particular be performed in the presence of abiocatalyst, catalysing this reaction step, in particular a biocatalystthat is capable of catalysing the reduction of an oxo group, inparticular a carbonyl group to a hydroxy group.

In a specific embodiment, the 3-oxoadipate is present as its thioesterwith co-enzyme A (hereinafter, the thioester of 3-oxoadipate andco-enzyme A will be referred to as 3-oxoadipyl-CoA).

In a preferred method of the invention, the preparation comprises abiocatalytic reaction in the presence of a biocatalyst capable ofcatalysing the reduction of a 3-oxoacyl (ester or thioester) to a3-hydroxyacyl (ester or thioester).

An enzyme having such catalytic activity may therefore be referred to asa 3-hydroxyacyl (ester or thioester) dehydrogenase. An enzyme havingsuch catalytic activity toward the 3-hydroxyacyl CoA-thioester maytherefore be referred to as a 3-hydroxyacyl-CoA dehydrogenase.Preferably, the said 3-hydroxyacyl-CoA dehydrogenase is selectivetowards the substrate 3-oxoadipyl-CoA.

An enzyme capable of catalysing the reduction of 3-oxoacyl (ester orthioester) to a 3-hydroxyacyl (ester or thioester) may in particular beselected from the group of dehydrogenases (E.C. 1.1.1), preferably fromthe group 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35 and EC 1.1.1.36),3-hydroxybutanoyl-CoA dehydrogenase (EC 1.1.1.157),3-hydroxypimeloyl-CoA dehydrogenase (EC 1.1.1.259) andlong-chain-3-hydroxyacyl-CoA dehydrogenases (EC 1.1.1.211). The enzymesmay use NADH or NADPH as cofactor. 3-Hydroxyacyl-CoA dehydrogenaseactivity has been described, for example, in fatty acid metabolism,polyketide biosynthesis, polyhydroxyalkanoate metabolism, butanoatemetabolism, as well as degradation of aromatic compounds according tothe KEGG (Kyoto Encyclopedia of Genes and Genomes) database.

In particular, microorganisms that are able to degrade an organiccompound, as identified above (see ‘reaction 1’), in particular anaromatic compound, an alicyclic compound or a dicarboxylic acid.

Other preferred organisms for providing a biocatalyst capable ofcatalysing the preparation of 3-hydroxyadipate (ester or thioester)include: Acinetobacter (in particular Acinetobacter sp. Strain ADP1 andA. calcoaceticus), Alicaligenes (in particular Alicaligenes strain D2and A. eutrophus), Arzoarcus (in particular A. evansii), Bacillus (inparticular B. halodurans), Bordetella (in particular B. pertussis),Burkholderia (in particular B. pseudomallei and B. xenovorans),Corynebacterium (in particular C. glutamicum, C. aurantiacum and C.efficiens), Deinococcus (in particular D. radiodurans), E. coli,Flavobacterium, Klebsiella (in particular K. pneumonia), Pseudomonas (inparticular P. putida and P. fluorescens), Rhodopseudomonas (inparticular R. palustris), Rodococcus (in particular R. erythropolis, R.opacus, and Rodococcus sp strain RHA1), Aspergillus (in particular A.niger), Neurospora (in particular N. crassa), Penicillium (in particularP. chrysogenum), Saccharomyces (in particular S. cerevisiae).

A suitable organism for providing an enzyme of EC1.1.1.35, acting on3-ketohexanoyl-CoA may be from any organism including mammals, inparticular mammals selected from the group of Bos taurus, Rattusnorvegicus, Sus scrofa, and Homo sapiens.

At suitable biocatalyst involved in (anaerobic) fatty acid synthesis,polyketide biosynthesis or polyhydroxyalkanoate metabolism may be fromany organism, in particular microorganisms including: Clostridia (inparticular C. acetobutylicum and C. kluyveri), Euglenozoa (in particularEuglena gracilis), Megasphera (in particular Megasphera elsdenii),Ralstonia (in particular Ralstonia eutropha), and Zoogloea (inparticular Zoogloea ramigera).

In a specific embodiment, the biocatalyst (catalysing ‘reaction 2’)comprises an enzyme comprising an amino acid sequence as identified inany of the SEQUENCE ID's 15-26, 29 or a homologue thereof. It isenvisaged that in particular an enzyme comprising an amino acid sequenceaccording to SEQUENCE ID 26 may catalyse both ‘reaction 2’ and ‘reaction3’.

The Preparation of 2,3-Dehydro Adipate (Ester or Thioester) Reaction 3

In an embodiment, 2,3-dehydro adipate (5-carboxy-2-pentenoate) (ester orthioester) is prepared from 3-hydroxyadipate (ester or thioester).Optionally, the 2,3-dehydro adipate and the 3-hydroxyadipate are coupledto a co-enzyme, ACP or another activating group, as indicated above.

In an embodiment of the invention, the 2,3-dehydro adipate (ester orthioester) is prepared by converting 3-hydroxyadipate (ester orthioester) chemically, e.g. by dehydration in a water free environmentin the presence of e.g. sulphuric acid.

The 2,3-dehydro adipate may in particular be prepared from3-hydroxyadipate using at least one biocatalyst.

A preferred biocatalyst is a biocatalyst that is capable of catalysingthe dehydration of a 3-hydroxyacyl ester or thioester to a 2-enoyl esteror 2-enoyl thioester thereof.

In a specific embodiment, the 2,3-dehydro adipate is present as itsthioester with co-enzyme A (hereinafter, the thioester of 2,3-dehydroadipate and co-enzyme A will be referred to as5-carboxy-2-pentenoyl-CoA).

In a specific embodiment, the biocatalyst catalyses the dehydration of3-hydroxyadipyl-CoA to 5-carboxy-2-pentenoyl-CoA.

In particular, the biocatalytic reaction may be carried out in thepresence of a biocatalyst capable of catalysing the dehydration of a3-hydroxyacyl (thio)ester to a 2,3-dehydroacyl thioester.

An enzyme having such catalytic activity may therefore be referred to asa 3-hydroxyacyl (ester or thioester) dehydratase. An enzyme having suchcatalytic activity toward the 3-hydroxyacyl CoA-thioester may thereforebe referred to as a 3-hydroxyacyl-CoA dehydratase. Preferably, the said3-hydroxyacyl-CoA dehydratase is selective towards the substrate3-hydroxyadipyl-CoA.

An enzyme capable of catalysing the dehydration of 3-hydroxyacyl (esteror thioester) to a 2,3-dehydroacyl (ester or thioester) may inparticular be selected from the group of hydrolyases (E.C. 4.2.1),preferably from the group of preferably from the group of enoyl-CoAhydratases (EC 4.2.1.17), 3-hydroxybutyryl-CoA dehydratases (EC4.2.1.55) and long-chain-enoyl-CoA hydratases (EC 4.2.1.74).3-Hydroxyacyl-CoA dehydratase activity has been described, for example,in fatty acid metabolism, polyketide synthesis, or butanoate metabolism,as well as degradation of aromatic compounds according to the KEGGdatabase.

A 3-hydroxyacyl (ester or thioester) dehydratase may be a 3-hydroxyacyl(ester or thioester) dehydratase of an organism selected from the groupof archaea, bacteria, and eukaryotes, for instance from the group ofyeasts, fungi and mammals.

In particular, microorganisms that are able to degrade an organiccompound, as identified above (see ‘reaction 1’), in particular anaromatic compound, an alicyclic compound or a dicarboxylic acid, arepreferred sources for a biocatalyst catalysing the preparation of2,3-dehydro adipate (ester or thioester).

Microorganisms capable of degrading an aromatic compound, an alicycliccompound or a dicarboxylic acid include Acinetobacter (in particularAcinetobacter sp. strain ADP1 and A. calcoaceticus), Alicaligenes (inparticular Alicaligenes D2), Aspergillus (in particular A. niger),Azoarcus (in particular A. evansii), Bacillus (in particular B.halodurans), Corynebacterium (in particular C. glutamicum and C.aurantiacum), E. coli, Flavobacterium, Neurospora (in particular N.crassa), Penicillium (in particular P. chrysogenum), Pseudomonas (inparticular P. putida and P. fluorescens), Rhodopseudomonas (inparticular R. palustris), Rhodococcus (in particular Rhodococcus spstrain RHA1).

A preferred organism for providing an enzyme of EC4.2.1.17, acting on3-hydroxyhexanoyl-CoA includes an organism selected from the group ofmammals and microorganisms. A suitable enzyme of EC4.2.1.17 from amammal may in particular be from a mammal selected from the group of Bostaurus, Homo sapiens, Rattus norvegicus, and Sus scrofa. A suitableenzyme of EC4.2.1.17 from a microorganism may in particular be from amicroorganism selected from the group of Aeromonas (in particular A.caviae), Clostridium (in particular C. acetobutylicumi), Gossypium (inparticular G. hirsutum), Rhodospirillum (in particular R. rubrumi), andRalstonia (in particular Ralstonia eutropha).

Preferred also are microorganisms capable of (anaerobic) fatty acidbiosynthesis. Such micro-organisms include Clostridia, in particular (C.acetobutylicum and C. kluyveri), Euglenozoa (in particular Euglenagracilis, Megasphera (in particular M. elsdenii), and Saccharomyces (inparticular S. cerevisiae).

A suitable enzyme may in particular comprise an amino acid sequenceaccording to any of the SEQUENCE ID's 14, 27, 28, 30-41, 92, or ahomologue thereof

The Preparation of Adipate (Ester or Thioester) from 2,3-Dehydro Adipate(Ester or Thioester) Reaction 4

In an embodiment, adipate (ester or thioester) is prepared from2,3-dehydro adipate (ester or thioester). Adipate (ester or thioester)may be prepared chemically from 2,3-dehydro adipate (ester orthioester), e.g. by selective hydrogenation of the C₂-C₃ doublebond, orbiocatalytically.

Usually, the 2,3-dehydro adipate is provided with an activating group,as indicated above.

The adipate (ester or thioester) preferably is prepared from 2,3-dehydroadipate (ester or thioester) using at least one biocatalyst, catalysingthe hydrogenation of the carbon-carbon double bond of5-carboxy-2-pentenoate (ester or thioester).

In a preferred method of the invention, the preparation comprises abiocatalytic reaction in the presence of a biocatalyst capable ofcatalysing the reduction of a cis or a trans 2-enoyl (ester orthioester) to an acyl (ester or thioester). The biocatalyst may use arange of electron donors, for example, an electron donor selected fromthe group of NADH, NADPH, FADH₂ and reduced ferredoxin. The electronsmay be transferred directly from the electron donor to the biocatalyst,or, alternatively, mediated, in particular by the so-called electrontransfer flavoprotein (ETF). An enzyme having such catalytic activitymay therefore be referred to as a 2-enoyl (ester or thioester) reductase(ER). An enzyme having such catalytic activity toward the 2-enoylCoA-thioester may therefore be referred to as a 2-enoyl-CoA reductase.Preferably, the said 2-enoyl-CoA reductase is selective towards thesubstrate 2,3-dehydroadipyl-CoA.

An enzyme capable of catalysing the reduction of 2-enoyl (ester orthioester) may in particular be selected from the group ofoxidoreductases (EC 1.3.1 and EC 1.3.99), preferably from the group ofenoyl-CoA reductases EC 1.3.1.8, EC 1.3.1.38 and EC 1.3.1.44, from thegroup of enoyl-[acyl-carrier-protein] reductases EC 1.3.1.9, EC 1.3.1.10and EC 1.3.1.39, and from the group butyryl-CoA dehydrogenase (EC1.3.99.2), acyl-CoA dehydrogenase (1.3.99.3) and long-chain-acyl-CoAdehydrogenase (EC 1.3.99.13). Trans-2-enoyl (ester or thioester)reductase activity has been described, for example, in fatty acidmetabolism, polyketide synthesis, butanoate metabolism and mitochondrialfatty acid biosynthesis according to the KEGG database.

An 2-enoyl (ester or thioester) reductase may in principle be obtainedor derived from any organism. In particular the organism can be selectedfrom bacteria, archaea, or eukariotes, such as from the group of yeasts,fungi, protists, plants and animals (including human).

In an embodiment, the organism may be selected from the followingbacteria: E. coli, Vibrio, Bacillus (in particular B. subtilis),Clostridia (in particular C. kluyveri, C. acetobutylicum, C.beijerinckii and C. perfringens), Streptomyces (in particular S.coelicolor and S. avermitilis), Pseudomonas (in particular P. putida andP. aeruginosa), Shewanella, Xanthomonas, Xylella, Yersinia, Treponema(in particular T. denticola), Aeromonas (in particular Aeromonashydrophila), Microscilla (in particular Microscilla marina), Megasphera(in particular Megasphera elsdenii), Deinococcus (in particularDeinococcus radiourans), Yarrowia (in particular Y. lypolytica) andEubacterium (in particular E. pyruvativorans).

In an embodiment an 2-enoyl (ester or thioester) reductase is from anorganism selected from the group of Euglenozoa, in particular Euglenagracilis)

In an embodiment an 2-enoyl (ester or thioester) reductase is from anorganism selected from the group of Saccharomyces (in particular S.cerevisiae), Kluyveromyces (in particular K. lactis),Schizosaccharomyces (in particular S. pombe), Candida (in particular C.tropicalis)

In an embodiment an 2-enoyl (ester or thioester) reductase is from anorganism selected from the group of Aspergillus (in particular A. nigerand A. nidulans), and Penicillium (in particular P. chrysogenum).

In an embodiment an 2-enoyl (ester or thioester) reductase is from anorganism selected from the group of Arabidopsis (in particular A.thaliana).

In an embodiment an 2-enoyl (ester or thioester) reductase is from anorganism selected from the group of Homo sapiens, Rattus norvegicus, BosTaurus, Cavia sp., Caenorhabditis elegans, and Drosophila melanogaster.

A suitable enzyme may in particular comprise an amino acid sequenceaccording to any of the SEQUENCE ID's 42-67, 94, 96, 98, 100, 105, 107,109, 111, 113, or a homologue thereof, in particular an amino acidsequence according to any of the SEQUENCE ID's 60, 63, 96, 100 or ahomologue thereof. Exemplary nucleotide sequences encoding a suitableenzyme for catalysing ‘reaction 4’ are represented by 2-enoyl (ester orthioester) reductase 93, 95, 97, 99, 104, 106, 108, 110 and 112.

In an advantageous embodiment, in addition to the 2-enoyl (ester orthioester) reductase an ETF is used, which may be beneficial to theactivity of said reductase. Such ETF may be obtained or derived from anorganism from which a reductase can be obtained or derived, asidentified above. In particular it may be obtained or derived from anorganism of the same genus, more in particular of the same species, asthe reductase that is used. Specific ETF's comprise a amino acidsequences represented by SEQUENCE ID's 102, 103, 115, 116. SEQUENCE ID's101 and 114 represent nucleotide sequences encoding a specific ETF.Usually, such ETFs comprise two subunits (etfA and etfB) encoded by twodifferent genes. These are generally used together to make the ETFprotein active. E.g. the following combinations could be used: SequenceID 102 with Sequence ID 103 or Sequence ID 116 with Sequence ID 115. Theskilled person will be able to selected other suitable ETF combinations,known in the art per se.

In an embodiment of the invention a biocatalyst not per se having adesired activity or substrate specificity may be modified by methodsknown in the art, e.g. by rational design or molecular evolution tocreate mutants able to catalyse the conversion of 2,3-dehydro adipate(ester or thioester) to adipate (ester or thioester) at a desirable rateor selectivity. Biocatalysts having activity with 2-enoyl-CoAderivatives with a chain length of 6, in particular such biocatalystsfrom C. kluyveri, Bos taurus, Euglena gracilis, Cavia sp., S.cerevisiae, C. tropicalis, Homo sapiens, and E. pyruvativorans arepreferred.

The Preparation of Adipic Acid Reaction 7

In accordance with the invention an adipate ester or thioester may beused to prepare adipic acid, by hydrolysis of the ester or thioesterbond. This may be accomplished chemically, e.g. by chemical hydrolysisin the presence of acid or base or biocatalytically.

In a preferred method of the invention, the preparation comprises abiocatalytic reaction in the presence of a biocatalyst capable ofcatalysing the hydrolysis of an acyl (thio)ester.

An enzyme having such catalytic activity may therefore be referred to asan acyl (thio)ester hydrolase. An enzyme having such catalytic activitytoward the acyl-CoA thioester may therefore be referred to as anacyl-CoA hydrolase. Preferably, the said acyl-CoA hydrolase is selectivetowards the substrate adipyl-CoA.

An enzyme capable of catalysing the hydrolysing an acyl (thio)ester mayin particular be selected from the group of hydrolases (EC 3.1.2),preferably from the group of acyl-CoA hydrolase (EC 3.1.2.20),acetyl-CoA hydrolase (EC 3.1.2.1), long-chain fatty-acyl-CoA hydrolase(EC 3.1.2.2), succinyl-CoA hydrolase (EC 3.1.2.3) andacyl-[acyl-carrier-protein]-hydrolase (EC 3.1.2.14).

The biocatalyst may comprise an enzyme originating from any organism,including archaea, bacteria or eukaryotes.

In particular, the biocatalyst may comprise an enzyme of a bacteriumselected from the group of E. coli, Brucella, (in particular Brucellamelitensis), Agrobacterium (in particular A. tumefaciens), Xanthomonas,Sinorhizobium (in particular Sinorhizobium meliloti), Mesorhizobium (inparticular Mesorhizobium loti), Vibrio, Streptomyces (in particular S.coelicolor and S. avermitilis), Rhodopseudomonas (in particularRhodopseudomonas palustris), Xylella, Yersinia, Pseudomonas (inparticular P. putida and P. aeruginosa), Shewanella, Shigella,Salmonella, Corynebacterium, Mycobacterium, Hyphomonas (in particularHyphomonas neptunium) and Propionibacterium.

A suitable biocatalyst may in particular be found in a yeast selectedfrom the group of Saccharomyces (in particular Saccharomyces cerevisiae)and Kluyveromyces (in particular K. lactis).

A suitable biocatalyst may in particular be found in a fungus selectedfrom the group of Aspergillus (in particular A. niger, A. fumigatus andA. nidulans) and Penicillium (in particular P. chrysogenum).

In a further embodiment, the organism is selected from the group ofArabidopsis (in particular A. thaliana), Muridae (in particular Rattusnorvegicus, Mus musculus), Bovidae (in particular Bos taurus, Ovisaries), Homo sapiens, and Caenorhabditis (in particular Caenorhabditiselegans).

In an embodiment of the invention a biocatalysts not per se having thedesired activity or substrate specificity may be modified by methodsknown in the art, e.g. by rational design or molecular evolution, tocreate mutants able to efficiently convert an adipate ester or thioesterto adipate. A biocatalyst having initial activity with a acyl-CoAderivative of a C4-C8 acid, preferably including dicarboxylic acids, arepreferred. For instance a mutant may be created based on anacyl-CoA-thioesterase from Mus musculus (e.g. as given in Seq ID 73).

In a specific embodiment of the invention, the preparation comprises abiocatalytic reaction in the presence of a biocatalyst capable ofcatalysing the transfer of an activating group, in particular an esteror thio-ester, most particular CoA.

An enzyme having such catalytic activity may therefore be referred to asa CoA transferase. Preferably, the said CoA transferase is selectivetowards a dicarboxylic-CoA as CoA-donating substrate. More preferably,the said dicarboxylic-CoA is adipyl-CoA. Preferably, the said CoAtransferase is selective towards or acetate as the CoA-acceptingsubstrate.

An enzyme capable of catalysing the transfer of a CoA group may inparticular be selected from the group of CoA transferases (EC 2.8.3),preferably from the group of dicarboxylic acid-CoA:dicarboxylic acid CoAtransferase, adipate:succinyl-CoA CoA transferase, 3-oxoacidCoA-transferase (EC 2.8.3.5), 3-oxoadipate CoA-transferase (EC 2.8.3.6)and acetate CoA-transferase (EC 2.8.3.8).

A CoA transferase may in principle be obtained or derived from anyorganism. The organism may be bacteria, archaea or eukaryotes. Inparticular, organisms that are able to degrade dicarboxylic acids, inparticular adipic acid, are preferred.

The organism may in particular be a bacterium selected from the group ofAcinetobacter (in particular Acinetobacter strain ADP1, A.calcoaceticus), Clostridium (in particular C. kluyveri, C.acetobutylicum or C. beijerinckii), Pseudomonas (in particular P. putidaand P. fluorescens), Agrobacterium, Alcaligenes, Athrobacter, Azomonas,Azospirillum, Azotobacter, Bacillus, Beijerinckia, Bradyrhizobium,Burkholderia, Comamonas, Corynebacterium, Norcadia, Rhizobium,Rhodotorula, Rodococcus, Trichosporon, and Roseburia sp.,

The organism may in particular be a yeast or fungus selected from thegroup of Aspergillus (in particular A. niger), Penicillium (inparticular P. chrysogenum), and Neurospora.

In particular, a suitable CoA transferase may be obtained or derivedfrom a species from the family of Hominidea, more in particular fromHomo sapiens.

A suitable enzyme for reaction 7 may in particular comprise an aminoacid sequence according to any of the SEQUENCE ID's 68-73, 85, 116, 117,119-124 or a homologue thereof.

The preparation of adipic acid from a thioester may in particular becatalysed by a biocatalyst comprising an acyl-CoA hydrolase comprisingan amino acid sequence according to any of the SEQUENCE ID's 68-73, 117,119 or a homologue thereof.

The preparation of adipic acid from a thioester may in particular becatalysed by a biocatalyst comprising a CoA transferase comprising anamino acid sequence according to any of the SEQUENCE ID's 85, 121, 122,123, 124, 125, 126 or a homologue thereof.

The CoA-transferase can be encoded by a single gene or by more than onegene. For instance, some CoA-transferases comprise two subunits encodedby two different genes. These are generally used together to make theCoA transferase protein active. E.g. the following combinations could beused: Sequence ID 121 with Sequence ID 122, or Sequence ID 125 withSequence ID 126.

The Preparation of 5-FVA from an Adipate Ester or an Adipate ThioesterReaction 5

In an embodiment, 5-formylpentanoate (5-FVA) is prepared from an adipateester or an adipate thioester. This may be done chemically, orbiocatalytically. The adipate may in particular be coupled to CoA oranother activating group, as indicated above.

In particular, the present invention also provides a method forpreparing 5-FVA from an adipate ester or an adipate thioester, inparticular a method for preparing 5-FVA from adipyl-CoA thioester, inthe presence of a biocatalyst capable of catalysing the reduction of anacyl ester or thioester to an aldehyde.

An enzyme having such catalytic activity may therefore be referred to asan aldehyde dehydrogenase. An enzyme having such catalytic activitytoward an acyl ester or acyl thioester—for instance acyl-CoAthioester—may therefore be referred to as an aldehyde dehydrogenase(acetylating). Preferably, the biocatalyst—comprising an aldehydedehydrogenase (acetylating)—is selective towards the substrateadipate-ester or thioester.

An enzyme capable of catalysing the reduction of an acyl (thio)ester mayin particular be selected from the group of oxidoreductases (EC 1.2.1),preferably from the group of aldehyde dehydrogenases (acetylating) (EC1.2.1.10), fatty acyl-CoA reductases (EC 1.2.1.42),long-chain-fatty-acyl-CoA reductases (EC 1.2.1.50), butanaldehydrogenases (EC 1.2.1.57) and succinate semialdehyde dehydrogenases(acetylating) (see e.g. Sohling et al. 1996. J. Bacteriol. 178: 871-880)

An aldehyde dehydrogenase may in principle be obtained or derived fromany organism. It is understood that the enzyme can also be obtained frommetagenomic sources by direct isolation of the encoding nucleic acid andsubsequent determination of activity in a heterologous host or bysequence homology found in the metagenomic DNA. The organism may bebacteria, archaea or eukaryotes. In particular the organism can beselected from bacteria, more in particular amongst the group of E. coli,Clostridium (in particular C. kluyveri, C. beijerinckii, C.acetobutylicum, C. botylicum, C. tetani, C. perfringens and C. novyi),Porphyromonas gingivalis, Listeria, Propionibacterium (in particular P.freudenreichii), Enterococcus, Fusobacterium, Lactobacillus (inparticular L. lactis), Bacillus (in particular B. thuringiensis),Burkholderia (in particular B. thailandensis and B. mallei), Pseudomonas(in particular P. putida), Rhodococcus (in particular R. sp. RHA1) andSalmonella (in particular S. typhimurium). The organism can also beselected from eukaryotes, more in particular amongst the group ofGiardia (in particular G. lamblia), Entamoeba (in particular E.Histolytica), Mastigamoeba balamuthi, Chlamydomonas reinhardtii,Polytomella, Piromyces, Cryptosporidium, and Spironucleus barkhanus.

A suitable dehydrogenase, may in particular comprise an amino acidsequence according to any of the SEQUENCE ID's 74-81, 139-148, or ahomologue thereof.

In an embodiment of the invention a biocatalyst not per se having thedesired activity or substrate specificity may be modified by methodsknown in the art, e.g. by rational design or molecular evolution, tocreate mutants able to convert adipate ester or thioester to 5-FVA.Biocatalysts having acylating aldehyde dehydrogenase activity withacyl-CoA derivatives with a chain length of 4-8, including but notlimited to biocatalysts such as succinate semialdehyde dehydrogenase(acetylating) from C. kluyveri (Sequence ID 74) or P. gingivialis(Sequence ID 75) and butylaldehyde dehydrogenase (acetylating) from C.acetobutylicum (Sequence ID 80, 81) or Propionibacterium freudenreichii(Sequence ID 79) are preferred.

The Preparation of 5-FVA from Adipic Acid Reaction 8

In accordance with the invention adipic acid may be used to prepare5-FVA, by reduction of one of the carboxylic acid groups. This may beaccomplished chemically, e.g. by selective chemical reduction optionallyincluding protection of one carboxylic acid group or biocatalytically.In a preferred method of the invention, the preparation comprises abiocatalytic reaction in the presence of a biocatalyst capable ofcatalysing the reduction a carboxylic acid. The biocatalyst may use NADHor NADPH as electron donor.

An enzyme having such catalytic activity may therefore be referred to asan aldehyde dehydrogenase. Preferably, the said aldehyde dehydrogenaseis selective towards the substrate adipate.

An enzyme capable of catalysing the reduction of a carboxylic acid mayin particular be selected from the group of oxidoreductases (EC 1.2.1),preferably from the group of aldehyde dehydrogenase (EC 1.2.1.3, EC1.2.1.4 and EC 1.2.1.5), malonate-semialdehyde dehydrogenase (EC1.2.1.15), succinate-semialdehyde dehydrogenase (EC 1.2.1.16 and EC1.2.1.24); glutarate-semialdehyde dehydrogenase (EC 1.2.1.20),aminoadipate semialdehyde dehydrogenase (EC 1.2.1.31), adipatesemialdehyde dehydrogenase (EC 1.2.1.63), which may also be referred toas 6-oxohexanoate dehydrogenase. Adipate semialdehyde dehydrogenaseactivity has been described, for example, in the caprolactam degradationpathway in the KEGG database. In particular a 6-oxohexanoatedehydrogenase may be used. Examples of 6-oxohexanoate dehydrogenases areenzymes comprising a sequence as represented by SEQUENCE ID 127, 128 ora homologue thereof.

An aldehyde dehydrogenase may in principle be obtained or derived fromany organism. The organism may be prokaryotic or eukaryotic. Inparticular the organism can be selected from bacteria, archaea, yeasts,fungi, protists, plants and animals (including human).

In an embodiment the bacterium is selected from the group ofAcinetobacter (in particular Acinetobacter sp. NCIMB9871), Ralstonia,Bordetella, Burkholderia, Methylobacterium, Xanthobacter, Sinorhizobium,Rhizobium, Nitrobacter, Brucella (in particular B. melitensis),Pseudomonas, Agrobacterium (in particular Agrobacterium tumefaciens),Bacillus, Listeria, Alcaligenes, Corynebacterium, and Flavobacterium.

In an embodiment the organism is selected from the group of yeasts andfungi, in particular from the group of Aspergillus (in particular A.niger and A. nidulans) and Penicillium (in particular P. chrysogenum)

In an embodiment, the organism is a plant, in particular Arabidopsis,more in particular A. thaliana.

The Preparation of 6-ACA Reaction 6

In an embodiment of the invention, 5-FVA is used to prepare 6-ACA.

In an embodiment, 6-ACA is obtained by hydrogenation over PtO₂ of6-oximocaproic acid, prepared by reaction of 5-FVA and hydroxylamine.(see e.g. F. O. Ayorinde, E. Y. Nana, P. D. Nicely, A. S. Woods, E. O.Price, C. P. Nwaonicha J. Am. Oil Chem. Soc. 1997, 74, 531-538 forsynthesis of the homologous 12-aminododecanoic acid).

6-ACA can be prepared in high yield by reductive amination of 5-FVA withammonia over a hydrogenation catalyst, for example Ni on SiO2/Al203support, as described for 9-aminononanoic acid (9-aminopelargonic acid)and 12-aminododecanoic acid (12-aminolauric acid) in EP-A 628 535 or DE4 322 065.

In a further embodiment 6-ACA is biocatalytically prepared. In apreferred method, the preparation of 6-ACA from 5-FVA comprises anenzymatic reaction in the presence of an enzyme capable of catalysing atransamination reaction in the presence of an amino donor, selected fromthe group of aminotransferases (E.C. 2.6.1).

In general, a suitable aminotransferase has 6-ACA 6-aminotransferaseactivity, capable of catalysing the conversion of 5-FVA into 6-ACA.

The aminotransferase may in particular be selected amongstaminotransferases from a mammal; Mercurialis, in particular Mercurialisperennis, more in particular shoots of Mercurialis perennis; Asplenium,more in particular Asplenium unilaterale or Asplenium septentrionale;Ceratonia, more in particular Ceratonia siliqua; Rhodobacter, inparticular Rhodobacter sphaeroides, Staphylococcus, in particularStaphylococcus aureus; Vibrio, in particular Vibrio fluvialis;Pseudomonas, in particular Pseudomonas aeruginosa; Rhodopseusomonas;Bacillus, in particular Bacillus weihenstephanensis and Bacillussubtilis; Legionella; Nitrosomas; Neisseria; or yeast, in particularSaccharomyces cerevisiae.

In case the enzyme is of a mammal, it may in particular originate frommammalian kidney, from mammalian liver, from mammalian heart or frommammalian brain. For instance a suitable enzyme may be selected amongstthe group of β-aminoisobutyrate:α-ketoglutarate aminotransferase frommammalian kidney, in particular β-aminoisobutyrate:α-ketoglutarateaminotransferase from hog kidney; β-alanine aminotransferase frommammalian liver, in particular β-alanine aminotransferase from rabbitliver; aspartate aminotransferase from mammalian heart; in particularaspartate aminotransferase from pig heart; 4-amino-butyrateaminotransferase from mammalian liver, in particular 4-amino-butyrateaminotransferase from pig liver; 4-amino-butyrate aminotransferase frommammalian brain, in particular 4-aminobutyrate aminotransferase fromhuman, pig, or rat brain; α-ketoadipate-glutamate aminotransferase fromNeurospora, in particular α-ketoadipate:glutamate aminotransferase fromNeurospora crassa; 4-amino-butyrate aminotransferase from E. coli, orα-aminoadipate aminotransferase from Thermus, in particularα-aminoadipate aminotransferase from Thermus thermophilus, and5-aminovalerate aminotransferase from Clostridium in particular fromClostridium aminovalericum. A suitable 2-aminoadipate aminotransferasemay e.g. be provided by Pyrobaculum islandicum.

In a specific embodiment, an aminotransferase is used comprising anamino acid sequence according to Sequence ID 82, Sequence ID 83,Sequence ID 84, Sequence ID 134, Sequence ID 136, Sequence 138, or ahomologue of any of these sequences. Sequence ID's 86 (wild-type) and 88(codon optimised) represent sequence encoding an enzyme represented bySequence ID 82 (=87). Sequence ID's 89 (wild-type) and 91 (codonoptimised) represent sequence encoding an enzyme represented by SequenceID 83 (=90). Sequence ID 133, Sequence ID 135, Sequence 137 representencoding sequences for Sequence ID 134, Sequence ID 136, Sequence 138,respectively.

In particular, the amino donor can be selected from the group ofammonia, ammonium ions, amines and amino acids. Suitable amines areprimary amines and secondary amines. The amino acid may have a D- orL-configuration. Examples of amino donors are alanine, glutamate,isopropylamine, 2-aminobutane, 2-aminoheptane, phenylmethanamine,1-phenyl-1-aminoethane, glutamine, tyrosine, phenylalanine, aspartate,β-aminoisobutyrate, β-alanine, 4-aminobutyrate, and α-aminoadipate.

In a further preferred embodiment, the method for preparing 6-ACAcomprises a biocatalytic reaction in the presence of an enzyme capableof catalysing a reductive amination reaction in the presence of anammonia source, selected from the group of oxidoreductases acting on theCH—NH₂ group of donors (EC 1.4), in particular from the group of aminoacid dehydrogenases (E.C. 1.4.1). In general, a suitable amino aciddehydrogenase has 6-aminocaproic acid 6-dehydrogenase activity,catalysing the conversion of 5-FVA into 6-ACA or has α-aminopimelate2-dehydrogenase activity, catalysing the conversion of AKP into AAP. Inparticular a suitable amino acid dehydrogenase be selected amongst thegroup of diaminopimelate dehydrogenases (EC 1.4.1.16), lysine6-dehydrogenases (EC 1.4.1.18), glutamate dehydrogenases (EC 1.4.1.3; EC1.4.1.4), and leucine dehydrogenases (EC 1.4.1.9).

In an embodiment, an amino acid dehydrogenase may be selected amongst anamino acid dehydrogenases classified as glutamate dehydrogenases actingwith NAD or NADP as acceptor (EC 1.4.1.3), glutamate dehydrogenasesacting with NADP as acceptor (EC 1.4.1.4), leucine dehydrogenases (EC1.4.1.9), diaminopimelate dehydrogenases (EC 1.4.1.16), and lysine6-dehydrogenases (EC 1.4.1.18).

An amino acid dehydrogenase may in particular originate from an organismselected from the group of Corynebacterium, in particularCorynebacterium glutamicum; Proteus, in particular Proteus vulgaris;Agrobacterium, in particular Agrobacterium tumefaciens; Geobacillus, inparticular Geobacillus stearothermophilus; Acinetobacter, in particularAcinetobacter sp. ADP1; Ralstonia, in particular Ralstonia solanacearum;Salmonella, in particular Salmonella typhimurium; Saccharomyces, inparticular Saccharomyces cerevisiae; Brevibacterium, in particularBrevibacterium flavum; and Bacillus, in particular Bacillus sphaericus,Bacillus cereus or Bacillus subtilis. For instance a suitable amino aciddehydrogenase may be selected amongst diaminopimelate dehydrogenasesfrom Bacillus, in particular Bacillus sphaericus; diaminopimelatedehydrogenases from Brevibacterium sp.; diaminopimelate dehydrogenasesfrom Corynebacterium, in particular diaminopimelate dehydrogenases fromCorynebacterium glutamicum; diaminopimelate dehydrogenases from Proteus,in particular diaminopimelate dehydrogenase from Proteus vulgaris;lysine 6-dehydrogenases from Agrobacterium, in particular Agrobacteriumtumefaciens, lysine 6-dehydrogenases from Geobacillus, in particularfrom Geobacillus stearothermophilus; glutamate dehydrogenases actingwith NADH or NADPH as cofactor (EC 1.4.1.3) from Acinetobacter, inparticular glutamate dehydrogenases from Acinetobacter sp. ADP1;glutamate dehydrogenases (EC 1.4.1.3) from Ralstonia, in particularglutamate dehydrogenases from Ralstonia solanacearum; glutamatedehydrogenases acting with NADPH as cofactor (EC 1.4.1.4) fromSalmonella, in particular glutamate dehydrogenases from Salmonellatyphimurium; glutamate dehydrogenases (EC 1.4.1.4) from Saccharomyces,in particular glutamate dehydrogenases from Saccharomyces cerevisiae;glutamate dehydrogenases (EC 1.4.1.4) from Brevibacterium, in particularglutamate dehydrogenases from Brevibacterium flavum; and leucinedehydrogenases from Bacillus, in particular leucine dehydrogenases fromBacillus cereus or Bacillus subtilis.

In an embodiment, 6-ACA prepared in a method of the invention is usedfor preparing caprolactam. Such method comprises cyclising the 6amino-caproic acid, optionally in the presence of a biocatalyst.

Reaction conditions for any biocatalytic step in the context of thepresent invention may be chosen depending upon known conditions for thebiocatalyst, in particular the enzyme, the information disclosed hereinand optionally some routine experimentation.

In principle, the pH of the reaction medium used may be chosen withinwide limits, as long as the biocatalyst is active under the pHconditions. Alkaline, neutral or acidic conditions may be used,depending on the biocatalyst and other factors. In case the methodincludes the use of a micro-organism, e.g. for expressing an enzymecatalysing a method of the invention, the pH is selected such that themicro-organism is capable of performing its intended function orfunctions. The pH may in particular be chosen within the range of fourpH units below neutral pH and two pH units above neutral pH, i.e.between pH 3 and pH 9 in case of an essentially aqueous system at 25° C.A system is considered aqueous if water is the only solvent or thepredominant solvent (>50 wt. %, in particular >90 wt. %, based on totalliquids), wherein e.g. a minor amount (<50 wt. %, in particular <10 wt.%, based on total liquids) of alcohol or another solvent may bedissolved (e.g. as a carbon source) in such a concentration thatmicro-organisms which may be present remain active. In particular incase a yeast and/or a fungus is used, acidic conditions may bepreferred, in particular the pH may be in the range of pH 3 to pH 8,based on an essentially aqueous system at 25° C. If desired, the pH maybe adjusted using an acid and/or a base or buffered with a suitablecombination of an acid and a base.

In principle, the incubation conditions can be chosen within wide limitsas long as the biocatalyst shows sufficient activity and/or growth. Thisincludes aerobic, micro-aerobic, oxygen limited and anaerobicconditions.

Anaerobic conditions are herein defined as conditions without any oxygenor in which substantially no oxygen is consumed by the biocatalyst, inparticular a micro-organism, and usually corresponds to an oxygenconsumption of less than 5 mmol/l.h, in particular to an oxygenconsumption of less than 2.5 mmol/l.h, or less than 1 mmol/l.h.

Aerobic conditions are conditions in which a sufficient level of oxygenfor unrestricted growth is dissolved in the medium, able to support arate of oxygen consumption of at least 10 mmol/l.h, more preferably morethan 20 mmol/l.h, even more preferably more than 50 mmol/l.h, and mostpreferably more than 100 mmol/l.h.

Oxygen-limited conditions are defined as conditions in which the oxygenconsumption is limited by the oxygen transfer from the gas to theliquid. The lower limit for oxygen-limited conditions is determined bythe upper limit for anaerobic conditions, i.e. usually at least 1mmol/l.h, and in particular at least 2.5 mmol/l.h, or at least 5mmol/l.h. The upper limit for oxygen-limited conditions is determined bythe lower limit for aerobic conditions, i.e. less than 100 mmol/l.h,less than 50 mmol/l.h, less than 20 mmol/l.h, or less than to 10mmol/l.h.

Whether conditions are aerobic, anaerobic or oxygen limited is dependenton the conditions under which the method is carried out, in particularby the amount and composition of ingoing gas flow, the actualmixing/mass transfer properties of the equipment used, the type ofmicro-organism used and the micro-organism density.

In principle, the temperature used is not critical, as long as thebiocatalyst, in particular the enzyme, shows substantial activity.Generally, the temperature may be at least 0° C., in particular at least15° C., more in particular at least 20° C. A desired maximum temperaturedepends upon the biocatalyst. In general such maximum temperature isknown in the art, e.g. indicated in a product data sheet in case of acommercially available biocatalyst, or can be determined routinely basedon common general knowledge and the information disclosed herein. Thetemperature is usually 90° C. or less, preferably 70° C. or less, inparticular 50° C. or less, more in particular or 40° C. or less.

Further, solvents, additional reagents and further aids, e.g. cofactors(for instance FAD/FADH and/or NAD/NADH cofactor) may be chosen based onknown reaction principles, to accomplish or accelerate a specificreaction and/or measures may be taken to shift the equilibrium to thedesired side. In particular if a biocatalytic reaction is performedoutside a host organism, a reaction medium comprising an organic solventmay be used in a high concentration (e.g. more than 50%, or more than 90wt. %), in case an enzyme is used that retains sufficient activity insuch a medium.

Succinate (ester or thioester) and acetate (ester or thioester) used ina method of the invention may in principle be obtained in any way.

Succinate is, e.g. naturally formed as an intermediate of the citricacid cycle (Krebs cycle) or an end product in cellular metabolism. Thus,it may be obtained from a renewable carbon source by using a suitablebiocatalyst. Biocatalysts, in particular microorganisms, can be used forproducing succinate from a suitable carbon source. The microorganism canbe a prokaryote or a eukaryote. The microorganism may be recombinant orwild type.

In a recombinant microorganism, the metabolism may be altered toincrease the yield and productivity of succinate on a suitable carbonsource. Methods for increasing succinate production have been describedfor prokaryotes in Song and Lee, Enzyme and Microbial Technology, 2006,39: 352-361. Succinate may also be produced in a eukaryote. In additionand alternatively, adpative evolutionary can be applied, such asdescribed in US application 2007/111294.

Succinate ester or thioester can be obtained from succinate in any way.In particular, succinate ester or thioester can be obtained fromsuccinate by using a biocatalyst. In particular, succinyl-CoA can beobtained from succinate by using a biocatalyst comprising an enzymeselected from the group of acid thiol ligase (EC 6.2.1), preferably fromthe group of succinyl-CoA synthase (EC 6.2.1.4 and EC 6.2.1.5). Inaddition or alternatively, succinyl-CoA can be obtained from succinateby using a biocatalyst comprising an enzyme selected from the group ofCoA transferases (EC 2.8.3) as specified for reaction 7.

Succinate ester or thioester can also be obtained from molecules otherthan succinate in any way. In particular, succinyl-CoA can be obtainedfrom 2-oxoglutarate using a biocatalyst comprising a 2-oxoglutaratedehydrogenase complex. 2-Oxoglutarate dehydrogenase complex is amulti-enzyme complex participating in the TCA cycle, known to personskilled in the art. In addition or alternatively, succinyl-CoA can beobtained from 2-oxoglutarate using a biocatalyst comprising a2-oxoglutarate:ferredoxin oxidoreductase (EC 1.2.7.3).

Acetate is a natural intermediate or end product in cellular metabolism.Thus, it may be obtained from a renewable carbon source by using asuitable biocatalyst. Biocatalysts, in particular microorganisms, can beused for producing succinate from a suitable carbon source. Themicroorganism can be a prokaryote or a eukaryote. The microorganism maybe recombinant or wild type.

Acetate ester or thioester can be obtained from acetate in any way. Inparticular, acetyl-CoA can be obtained from acetate by using abiocatalyst comprising an enzyme selected from the group of acid thiolligase (EC 6.2.1), preferably acetyl-CoA synthase (EC 6.2.1.1 and EC6.2.1.13). In addition or alternatively, acetyl-CoA can be obtained fromacetate using a biocatalyst comprising of an enzyme selected from thegroup of CoA transferases (EC 2.8.3) as specified for reaction 7.

Acetate ester or thioester can also be obtained from molecules otherthan acetate in any way. In particular, acetyl-CoA can be obtained frompyruvate using a biocatalyst comprising an enzyme selected from thegroup of pyruvate dehydrogenase complex, pyruvate dehydrogenase (NADP+)(EC 1.2.1.51), pyruvate formate lyase (EC 2.3.1.54) or a biocatalyst orenzyme effectively converting pyruvate to acetyl-CoA. Pyruvatedehydrogenase complex is a multi-enzyme complex converting pyruvate intoacetyl-CoA, known to person skilled in the art.

Acetyl-CoA can also be obtained from acetaldehyde using a biocatalystcomprising an enzyme selected from the group of oxidoreductases (EC1.2.1), preferably from the group of aldehyde dehydrogenases(acetylating) (EC 1.2.1.10), fatty acyl-CoA reductases (EC 1.2.1.42),butanal dehydrogenases (EC 1.2.1.57) and succinate semialdehydedehydrogenases (acetylating) (as described in Sohling et al. 1996. J.Bacteriol. 178: 871-880).

When the biocatalyst is a eukaryote, the supply of acetyl-CoA,preferably in the cytosolic compartment in the host cell, may beincreased by overexpressing homologous and/or heterologous genesencoding enzymes that catalyze the conversion of a precursor molecule toacetyl-CoA. The precursor molecule may for example be acetate, asdescribed by Shiba et al., Metabolic Engineering, 2007, 9: 160-8.

In an advantageous method of the invention, in particular a method forpreparing 6-ACA, adipic acid or an intermediate compound for 6-ACA oradipic acid, use is made of a whole cell biotransformation of thesubstrate for 6-ACA, adipic acid or an intermediate thereof, comprisingthe use of a micro-organism wherein one or more enzymes catalysing anyof the above reactions are produced, and a carbon source for themicro-organism.

The carbon source may in particular contain at least one compoundselected from the group of monohydric alcohols, polyhydric alcohols,carboxylic acids, carbon dioxide, fatty acids, glycerides, includingmixtures comprising any of said compounds. Suitable monohydric alcoholsinclude methanol and ethanol, Suitable polyols include glycerol andcarbohydrates. Suitable fatty acids or glycerides may in particular beprovided in the form of an edible oil, preferably of plant origin.

In particular a carbohydrate may be used, because usually carbohydratescan be obtained in large amounts from a biologically renewable source,such as an agricultural product, for instance an agriculturalwaste-material. Preferably a carbohydrate is used selected from thegroup of glucose, fructose, sucrose, lactose, saccharose, starch,cellulose and hemi-cellulose. Particularly preferred are glucose,oligosaccharides comprising glucose and polysaccharides comprisingglucose.

In a specific method of the invention, the method is a fermentationmethod. Such method may in particular, comprise contacting cellscomprising a biocatalyst—optionally a host cell as described herein—witha fermentable carbon source, wherein the carbon source contains any ofsaid compounds which are to be converted into the compound to beprepared or wherein the cells prepare the compound to be converted intothe compound to be prepared from the carbon source.

A cell comprising one or more enzymes for catalysing a reaction step ina method of the invention can be constructed using molecular biologicaltechniques, which are known in the art per se. For instance, if one ormore biocatalysts are to be produced in a heterologous system, suchtechniques can be used to provide a vector which comprises one or moregenes encoding one or more of said biocatalysts. A vector comprising oneor more of such genes can comprise one or more regulatory elements, e.g.one or more promoters, which may be operably linked to a gene encodingan biocatalyst.

As used herein, the term “operably linked” refers to a linkage ofpolynucleotide elements (or coding sequences or nucleic acid sequence)in a functional relationship. A nucleic acid sequence is “operablylinked” when it is placed into a functional relationship with anothernucleic acid sequence. For instance, a promoter or enhancer is operablylinked to a coding sequence if it affects the transcription of thecoding sequence.

As used herein, the term “promoter” refers to a nucleic acid fragmentthat functions to control the transcription of one or more genes,located upstream with respect to the direction of transcription of thetranscription initiation site of the gene, and is structurallyidentified by the presence of a binding site for DNA-dependent RNApolymerase, transcription initiation sites and any other DNA sequences,including, but not limited to transcription factor binding sites,repressor and activator protein binding sites, and any other sequencesof nucleotides known to one of skilled in the art to act directly orindirectly to regulate the amount of transcription from the promoter. A“constitutive” promoter is a promoter that is active under mostenvironmental and developmental conditions. An “inducible” promoter is apromoter that is active under environmental or developmental regulation.The term “homologous” when used to indicate the relation between a given(recombinant) nucleic acid or polypeptide molecule and a given hostorganism or host cell, is understood to mean that in nature the nucleicacid or polypeptide molecule is produced by a host cell or organisms ofthe same species, preferably of the same variety or strain.

The promoter that could be used to achieve the expression of thenucleotide sequences coding for an enzyme for use in a method of theinvention such as described herein above may be native to the nucleotidesequence coding for the enzyme to be expressed, or may be heterologousto the nucleotide sequence (coding sequence) to which it is operablylinked. Preferably, the promoter is homologous, i.e. endogenous to thehost cell.

If a heterologous promoter (to the nucleotide sequence encoding for theenzyme of interest) is used, the heterologous promoter is preferablycapable of producing a higher steady state level of the transcriptcomprising the coding sequence (or is capable of producing moretranscript molecules, i.e. mRNA molecules, per unit of time) than is thepromoter that is native to the coding sequence. Suitable promoters inthis context include both constitutive and inducible natural promotersas well as engineered promoters, which are well known to the personskilled in the art.

A “strong constitutive promoter” is one which causes mRNAs to beinitiated at high frequency compared to a native host cell. Examples ofsuch strong constitutive promoters in Gram-positive micro-organismsinclude SP01-26, SP01-15, veg, pyc (pyruvate carboxylase promoter), andamyE.

Examples of inducible promoters in Gram-positive micro-organismsinclude, the IPTG inducible Pspac promoter, the xylose inducible PxylApromoter.

Examples of constitutive and inducible promoters in Gram-negativemicroorganisms include, tac, tet, trp-tet, lpp, lac, lpp-lac, laclq, T7,T5, T3, gal, trc, ara (P_(BAD)), SP6, λ-P_(R), and λ-P_(L).

The term “heterologous” when used with respect to a nucleic acid (DNA orRNA) or protein refers to a nucleic acid or protein that does not occurnaturally as part of the organism, cell, genome or DNA or RNA sequencein which it is present, or that is found in a cell or location orlocations in the genome or DNA or RNA sequence that differ from that inwhich it is found in nature. Heterologous nucleic acids or proteins arenot endogenous to the cell into which it is introduced, but has beenobtained from another cell or synthetically or recombinantly produced.Generally, though not necessarily, such nucleic acids encode proteinsthat are not normally produced by the cell in which the DNA istranscribed or expressed. Similarly exogenous RNA encodes for proteinsnot normally expressed in the cell in which the exogenous RNA ispresent. Heterologous nucleic acids and proteins may also be referred toas foreign nucleic acids or proteins. Any nucleic acid or protein thatone of skill in the art would recognize as heterologous or foreign tothe cell in which it is expressed is herein encompassed by the termheterologous nucleic acid or protein.

A method according to the invention may be carried out using anorganism, which may be a host organism, in particular a hostmicro-organism, or a wild-type micro-organism. Accordingly, theinvention also relates to a novel (host) cell, which may be amicroorganism, comprising a biocatalyst capable of catalysing at leastone reaction step in a method of the invention, preferably the cell iscapable of producing an enzyme or a plurality of enzymes, whereby two ormore reaction steps in a method of the invention are catalysed. Theinvention also relates to a novel vector comprising one or more genesencoding for one or more enzymes capable of catalysing at least onereaction step in a method of the invention.

In an embodiment, a cell or a vector is provided comprising a nucleicacid sequence, which may be recombinant, encoding an enzyme with5-carboxy-2-pentenoyl ester or thioester hydrogenase activity, inparticular 5-carboxy-2-pentenoyl hydrogenase activity.

Preferably, the cell further comprises at least one (recombinant vectorcomprising a) nucleic acid sequence encoding an enzyme selected from thegroup of enzymes capable of catalysing the conversion of an adipyl esteror thioester, in particular adipyl-Coa, into 5-FVA and enzymescatalysing the conversion of adipyl ester or thioester, in particularadipyl-Coa, into adipic acid.

In particular in an embodiment wherein the cell comprises an enzymecapable of catalysing the conversion of an adipyl ester or thioesterinto 5-FVA, the cell may advantageously comprise (a recombinant vectorcomprising) a nucleic acid sequence encoding an enzyme capable ofcatalysing the conversion of 5-FVA into 6-ACA. Such enzyme may inparticular be an enzyme with 5-FVA aminotransferase activity.

In addition or alternatively, the (host) cell respectively vectorcomprises at least one of the following nucleic acid sequences:

-   -   a nucleic acid sequence encoding an enzyme capable of catalysing        the formation of 3-oxoadipyl ester or thioester by reacting a        succinyl ester or thioester with an acetate ester or thioester;    -   a nucleic acid sequence encoding an enzyme capable of catalysing        the formation of a 3-hydroxyadipyl ester or thioester from a        3-oxoadipyl ester or thioester;    -   a nucleic acid sequence encoding an enzyme capable of catalysing        the formation of a 5-carboxy-2-pentenoyl ester or thioester from        a 3-hydroxyadipyl ester or thioester;    -   a nucleic acid sequence encoding an enzyme capable of catalysing        the formation of a an adipyl ester or thioester from        5-carboxy-2-pentenoyl ester or thioester.

One or more suitable genes may in particular be selected amongst genesencoding an enzyme as mentioned herein above, more in particular amongstgenes encoding an enzyme according to any of the Sequence ID's 1-67, 94,96, 98, 100, 102, 103, 105, 107, 109, 111, 113, 115, 116 or a homologuethereof.

The host cell may be a prokaryote or an eukaryote. In particular thehost cell can be selected from bacteria, archaea, yeasts, fungi,protists, plants and animals (including human).

In particular a host cell according to the invention may be selectedfrom the group of genera consisting of Aspergillus, Bacillus,Corynebacterium, Escherichia, Saccharomyces, Pseudomonas, Gluconobacter,Penicillium, Pichia. In particular a host strain and, thus, a host cellmay be selected from the group of E. coli, Bacillus subtilis, Bacillusamyloliquefaciens, Corynebacterium glutamicum, Aspergillus niger,Penicillium chrysogenum, Pichia pastoris, Saccharomyces cerevisiae.

Host cells able to produce short chain fatty acids such as succinateand/or acetate and/or esters or thio-esters thereof may be advantageous.Organisms capable thereof are generally present in the rumen ofruminants. In particular an organism able of coproduction of succinateand acetate or esters or thioesters thereof is preferred.

The microorganism may be recombinant or wild type. In particular,microorganisms capable of producing succinate include E. coli,Actinobacillus (in particular A. succinogenes), Mannheimia (inparticular M. succiniciproducens), Saccharomyces cerevisiae, Aspergillus(in particular A. niger), Penicillium (in particular P. chrysogenum andP. simplicissimum) and other organisms mentioned in Kaemwich Jantama, M.J. Haupt, Spyros A. Svoronos, Xueli Zhang, J. C. Moore, K. T. Shanmugam,L. O. Ingram. Biotechnology and Bioengineering (2007) 99, 5: 1140-1153.

In particular, microorganisms capable of producing acetate includeEnterobacteriaceae (in particular E. coli, Salmonella, and Shigella),acetic acid bacteria (Includes Acetobacter (in particular Acetobacteraceti), Gluconobacter (in particular Gluconobacter oxidans), Acidomonas,Gluconacetobacter, Asaia, Kozakia, Swaminathania, Saccharibacter,Neoasaia, Granulibacter, Clostridium (in particular C. aceticum, C.thermoaceticum, C. thermoautotrophicum, C. formicoaceticum, C. kluyveri,C. propionicum), Megasphaera (in particular M. elsdenii), Acetobacterium(In particular A. woodii and A. wieringae), Lactobacillus (in particularL. plantarum, L. brevum), Bifidobacterium (In particular B. bifidum),and Leuconostoc.

The invention further relates to a novel polypeptide, respectively to anucleotide sequence encoding such polypeptide. In particular, theinvention further relates to a polypeptide comprising an amino acidsequence according to any of the Sequence ID's 57, 68-72, 79, 85 andhomologues thereof. In particular, the invention further relates to apolynucleotide encoding a polypeptide comprising an amino acid sequenceaccording to any of the Sequence ID's 57, 68-72, 79, 85 and homologuesthereof.

Next, the invention is illustrated by the following examples.

EXAMPLES Example 1 General Methods

Molecular and Genetic Techniques

Standard genetic and molecular biology techniques are generally known inthe art and have been previously described (Maniatis et al. 1982“Molecular cloning: a laboratory manual”. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.; Miller 1972 “Experiments in moleculargenetics”, Cold Spring Harbor Laboratory, Cold Spring Harbor; Sambrookand Russell 2001 “Molecular cloning: a laboratory manual” (3rd edition),Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press; F.Ausubel et al, eds., “Current protocols in molecular biology”, GreenPublishing and Wiley Interscience, New York 1987).

Plasmids and Strains

pBAD/Myc-His C and pET21d were obtained from Invitrogen (Carlsbad,Calif., USA) and EMD Biosciences (Darmstadt, Germany) respectively.pF113 (a derivative of pJF119EH (Fürste, J. P., W. Pansegrau, R. Frank,H. Blocker, P. Scholz, M. Bagdasarian, and E. Lanka. 1986. Molecularcloning of the plasmid RP4 primase region in a multi-host-range tacPexpression vector. Gene 48:119-131.) which contains two NotI sites atpositions 515 and 5176 respectively with the tac promoter being thestart of the numbering), pACYC-tac (Kramer, M. (2000). Untersuchungenzum Einfluss erhöhter Bereitstellung von Erythrose-4-Phosphat andPhosphoenolpyruvat auf den Kohlenstofffluss in denAromatenbiosyntheseweg von Escherichia coli. Berichte desForschungszentrums Jülich, 3824. ISSN 0944-2952 (PhD Thesis, Universityof Düsseldorf) and pMS470 (Balzer, D.; Ziegelin, G.; Pansegrau, W.;Kruft, V.; Lanka, E. Nucleic Acids Research 1992, 20(8), 1851-1858.)have been described previously. E. coli TOP10 (Invitrogen, Carlsbad,Calif., USA) was used for all cloning procedures. E. coli strains Top10(Invitrogen, Carlsbad, Calif., USA), Rv308 (ATCC31608), Rv308ΔaraB, andBL21 A1 (Invitrogen, Carlsbad, Calif., USA) were used for proteinexpression.

All vectors were adapted by inserting a common linker to allow anidentical cloning strategy. The adaptation and general cloning scheme isshown in FIG. 2.

Media

2×TY medium (16 g/l tryptopeptone, 10 g/l yeast extract, 5 g/l NaCl) wasused for growth of E. coli. Antibiotics (100 μg/ml ampicillin) weresupplemented to maintain plasmids. For induction of gene expressionarabinose (for pBAD derivatives), IPTG (for pMS470 and pF113derivatives), and a combination of arabinose and IPTG (for pET21dderivatives in E. coli BL21-A1) were used at 0.005-0.2% (arabinose) and0.1-0.5 mM (IPTG) final concentrations.

Identification of Plasmids

Plasmids carrying the different genes were identified by genetic,biochemical, and/or phenotypic means generally known in the art, such asresistance of transformants to antibiotics, PCR diagnostic analysis oftransformant or purification of plasmid DNA, restriction analysis of thepurified plasmid DNA or DNA sequence analysis.

HPLC-MS Analysis Method for the Determination of CoA-Derivatives

Adipyl-Coa and 6-carboxy-2,3-ene hexanoyl-CoA concentrations weredetermined by LC-MS. An Agilent SB-C18 2.1*50 mm column was used forseparation with acetonitrile/water buffered with 750 mg/l octylammoniumacetate (pH=7.5) as mobile phase. Flow was 300 μl/min and elution wasdone with a gradient (Start: 70% water, decrease to 58% in 3 min, stepto 45%, further decrease to 20% in 1.5 min, followed by reequilibrationof the column, in such a way that the total runtime was 7 min). A LTQorbitrap was used in electrospray negative ionization mode, scanningfrom m/z 765-900. adipyl-Coa and 6-carboxy-2,3-ene hexanoyl-CoA elutedat 2.25 min and 2.5 min respectively. The selectivity of the method wasenhanced by observing the accurate protonated molecules of the compoundsrequested (adipyl-Coa: 894.15123-894.16017, 6-carboxy-2,3-enehexanoyl-CoA: 892.13437-892.14329). To determine concentrations astandard curve of synthetically prepared compounds was run to calculatea response factor for the respective ions. This was used to calculatethe concentrations in unknown samples.

Adipate can be detected and quantified as described in Kippenberger, M.;Winterhalter, R.; Moortgat, G. K. Anal. Bioanal. Chem. 2008, 392(7-8),1459-1470.

Example 2 6-carboxy-2,3-ene-hexanoyl-CoA reductase activitydetermination

Expression Constructs

Putative 6-carboxy-2,3-ene-hexanoyl-CoA reductases were selected fromdatabases (Table 1).

Target genes encoding the selected proteins were codon pair optimized(using methodology described in WO08000632) and constructedsynthetically (Geneart, Regensburg, Germany). Before optimization,targeting sequences (e.g secretion signals or peroxisomal/mitochondrialtargeting sequences) were removed from the amino acid sequence. Suchtargeting sequences can be identified by bioinformatics tools well knownin the art, such as described in Emanuelsson et al. 2007. Natureprotocols, 2: 953-971). In the optimization procedure internalrestriction sites were avoided and common restriction sites wereintroduced at the start and stop to allow cloning according to thestrategy shown in FIG. 2. These modifications may result in minorchanges to the respective protein sequences which are contemplated tonot alter the properties of the respective protein in any way. Each ORFwas preceded by a consensus ribosomal binding site and leader sequenceto drive translation in pF113, pMS470 and pET21d. In pBAD translationinitiation signals are provided by the vector. The target genes ‘Adi4’,Adi5’, ‘Adi8’ and ‘Adi9’ were cloned into all four plasmids both withand without read-through to the C-terminal His-tag provided by thelinker sequence.

Protein Expression in E. coli

Starter cultures were grown overnight in 96-well plates with 200 μlmedium/well. 40-160 μl were transferred to fresh 24-deep-well plateswith 4 ml media. For pBAD constructs in E. coli TOP10 or E. coliRv308ΔaraB this medium directly contained 0.005% arabinose forinductions. Plates were incubated on an orbital shaker (Infors, 550 rpm)at 25° C. After 4-6 h inducers were added (0.5 mM IPTG for pF113 andpMS470 in E. coli Rv308, E. coli BL21 or E. coli TOP10; 0.5 mM IPTG and0.2% arabinose for pET21d in E. coli BL21A1) and plates were incubatedfor another 4-48 h until cells were collected by centrifugation.

Preparation of Cell Free Extract and His-Tag Purification

Cells from small scales growth (see previous paragraph) were harvestedby centrifugation and the supernatant was discarded. The cell pelletsformed during centrifugation were frozen at −20° C. for at least 16 hand then thawed on ice. 2 ml of freshly prepared lysis buffer (50 mMpotassium phosphate pH7.5, 0.1 mg/ml DNAse I (Roche, Almere, NL), 2mg/ml Lysozyme, 0.5 mM MgSO₄, 1 mM dithiothreitol, and proteaseinhibitors (Complete Mini EDTA-free tablets™, Roche, Almere, NL, wereused according to the manufacturers specification) were added to eachwell and cells were resuspended by vigorously vortexing the plate for2-5 min. To achieve lysis, the plate was incubated at room temperaturefor 30 min. To remove cell debris, the plate was centrifuged at 4° C.and 6000 g for 20 min. The supernatant was transferred to a fresh plateand kept on ice until further use. For purification of His-taggedproteins His Multitrap HP filter plates (GE Healthcare bioscience AB,Uppsala, Sweden) were used according to the manufacturer's instructions.

Synthesis of Substrates

Substrate (J. R. Stern, A. del Campillo, J. Biol. Chem., 1956, 985. A.K. Das, M. D. Uhler, A. K. Hajra, J. Biol. Chem., 2000, 24333. H. Oku,N. Futamori, K. Masuda, Y. Shimabukuro, T. Omine, H. Iwasaki, Biosc.Biotech. Biochem., 2003, 2107. Elvidge et. al, J. Chem. Soc., 1953,1793. F. Liu, H-Y. Zha, Z-J. Yao, J. Org. Chem., 2003, 6679-6684) andproduct (WO2004/106347) of the desired biochemical reaction and weresynthesized by Syncom (Groningen, NL) according to published procedures.

Enzymatic Enoyl-CoA Assay

A reaction mixture was prepared comprising 50 mM potassium buffer(pH7.5), 0.7 mM NADH and NADPH each, and approximately 20 μM of thesubstrate 6-carboxylic 2,3-ene hexanoyl-CoA. 190 μl of the reactionmixture were dispensed into each well of 96-wellplates. The same way 10μl of cell free extract prepared from the respective strain carrying theempty vector was used in control reactions. To start the reaction, 10 μlof the cell free extracts or purified protein were added, to each of thewells. Reaction mixtures were incubated at room temperature (20-25 C)for 15 min to 24 h with online monitoring of UV absorption at 340 nm. Atthe end reactions were stopped by adding an equal volume of MeOH andsamples were centrifuged. Supernatant was transferred to a fresh plateand stored at −80 C until further analysis by HPLC-MS. adipyl-Coa wasfound as shown in Table 1.

TABLE 1 adipyl-CoA (amount is indicated by relative peak-area) found inthe enzymatic assay. Biocatalyst SeqID # Modifications¹ adipyl-CoA² Adi463 N-terminus 134 AA 3266 removed Adi5 96 196031 Adi8 60 N-terminus 22AA 581859 removed Adi9 100 N-terminus 12 AA 117077 removed - (vector 0control) ¹If the resulting polypeptide after modification does not startwith a methionine at the N-terminus, the resulting polypeptide isfurther modified by adding a methionine at the N-terminus. ²Resultsshown were obtained with E. coli BL21 containing the adi gene cloned inpMS470 after 20 h incubation. Positive results were also obtained withother expression vectors and host strains (such as pET21d in E. coliBL21-A1, pBAD/Myc-His C in E. coli Rv308 and pF113 in E. coli BL21) anddifferent incubation periods (e.g. 2 h).

Example 3 Production of Adipate by a Heterologous Microorganism

Construction of an Adipate Biosynthetic Pathway

A synthetic pathway was designed consisting of the enzymatic activitiesshown in FIG. 3. Enzymes encoding these activities were identified indatabases. Target genes encoding these enzymes were codon pair optimizedand constructed synthetically (Geneart, Regensburg, Germany). In theoptimization procedure internal restriction sites, undesired targetingsequences (e.g secretion signals or peroxisomal targeting sequences)were removed and restriction sites were introduced at the start and stopto allow assembly of the pathway in expression vectors according to FIG.4. These modifications may result in minor changes to the proteinsequence which are contemplated to not alter the properties of therespective protein in any way. Each ORF was preceded by a consensusribosomal binding site and leader sequence to drive translation.

For reactions 1, 2, and 3 the combinations of adi21+22+23 or adi26+27+28were used. For reaction 7 adi29, adi30 or a combination of adi24+25 wereused. For reaction 4 adi1-20 can be used with adi8, adi6, adi13+12.

TABLE 2 SeqIDs of the different Adi proteins. Protein Seq ID # Adi21 5Adi22 18 Adi23 33 Adi24 119 Adi25 120 Adi26 3 Adi27 29 Adi28 14 Adi29116 Adi30 117

Construction of an Adipate Producing E. coli Strain

To construct adipate producing E. coli strains plasmids encoding acomplete adipate pathway were transformed into the appropriate hoststrains for expression of the cloned genes. pMS470 constructs containingfull pathways, were transformed into E. coli BL21, TOP10 and Rv308.pBAD/Myc-His C constructs were transformed into E. coli TOP10 andRv308ΔaraB. Constructs in pF113 or pACYC-tac were transformed togetherwith compatible pF113, pACYC-tac or pMS470 constructs in a way that thefinal strain contained a complete adipate pathway. These plasmids wereco-transformed to E. coli TOP10, BL21, and Rv308.

Production of Adipate

For production of adipate, starter cultures were grown over night in96-well plates with 200 μl medium at 30 C. 50 μl were transferred to afresh plate 24-well plate with 4 ml medium and grown for 4-6 h at 25 Cand then inducers to induce expression of the adipate pathway wereadded. Cultures were incubated for another 12 h-72 h at 25 C. Plateswere centrifuged and samples prepared for LC-MS analysis. Supernatantwas mixed 1:1 with MeOH to precipitate proteins and then directlyanalyzed. Metabolites from cells were extracted by resuspending thepellet in 1 ml Ethanol. The cell suspension was transferred to a tubewith a screw top and heated in a boiling water bath for 3 min. Aftercentrifugation the supernatant was transferred to a fresh tube andevaporated in a speed-vac. Dry samples were resuspended in 100 μl mobilephase prior to analysis.

Example 4 Preparation of 5-FVA from Adipyl-CoA

HPLC-MS Analysis Method for the Determination of 5-FVA

5-FVA was detected by selective reaction monitoring (SRM)-MS, measuringthe transition m/z 129→83. Concentrations for 5-FVA were calculated bymeasuring the peak area of the 5-FVA peak eluting at approximately 6min. Calibration was performed by using an external standard procedure.All the LC-MS experiments were performed on an Agilent 1200 LC system,consisting of a quaternary pump, autosampler and column oven, coupledwith an Agilent 6410 QQQ triple quadrupole MS.

-   LC conditions:-   Column: 50×4.6 mm Nucleosil C18, 5 μm (Machery & Nagel) pre column    coupled to a 250×4.6 mm id. Prevail C18, 5 μm (Alltech)-   Column temperature: room temperature-   Eluent:    -   A: water containing 0.1% formic acid    -   B: acetonitrile containing 0.1% formic acid-   Gradient:

time (min) % eluent B 0 10 6 50 6.1 10 11 10

-   Flow: 1.2 ml/min, before entering the MS the flow is split 1:3-   Injection volume: 2 μl-   MS conditions:-   Ionisation: negative ion electrospray    -   source conditions:        -   ionspray voltage: 5 kV        -   temperature: 350° C.        -   fragmentor voltage and collision energy optimized-   Scan mode: selective reaction mode: transition m/z 129→83

Expression Constructs

Putative adipyl-CoA-reductases were selected (Seq ID 74, 75, 77, 79, 80,139-148). Expression constructs are designed and prepared in the sameway as described before in example 2 using pBAD/Myc-His C and pET21d.

Protein Expression, Extraction and Purification

All steps are carried out as described in example 2.

Enzymatic Adipyl-CoA Reductase Assay

A reaction mixture is prepared comprising 50 mM potassium buffer(pH7.5), 0.7 mM NADH and NADPH each, and 10 μM-10 mM of the substrateadipyl-CoA. 190 μl of the reaction mixture are dispensed into each wellof 96-wellplates. Adipyl-CoA was prepared as described in example 2. Tostart the reaction, 10 μl of the cell free extracts or purified proteinare added to each of the wells. The same way 10 μl of cell free extractprepared from the respective strain carrying the empty vector is used incontrol reactions. Reaction mixtures are incubated at room temperature(20-25 C) for 15 min-24 h with online monitoring of UV absorption at 340nm. At the end reactions are stopped by adding an equal volume of MeOHand samples are centrifuged. Supernatant is transferred to a fresh plateand stored at −80 C until detection of 5-FVA by HPLC-MS. Measurement of5-FVA demonstrates adipyl-CoA reductase activity of the selectedenzymes.

Example 5 The Preparation of 6-ACA from 5-FVA

HPLC-MS Analysis for the Determination of 6-ACA

Calibration:

The calibration was performed by an external calibration line of 6-ACA(m/z 132→m/z 114, Rt 7.5 min). All the LC-MS experiments were performedon an Agilent 1100, equipped with a quaternary pump, degasser,autosampler, column oven, and a single-quadrupole MS (Agilent,Waldbronn, Germany). The LC-MS conditions were:

-   Column: 50*4 Nucleosil (Mancherey-Nagel)+250×4.6 Prevail C18    (Alltech), both at room temperature (RT)-   Eluent:    -   A=0.1 (v/v) formic acid in ultrapure water    -   B=Acetonitrile (pa, Merck)-   Flow: 1.0 ml/min, before entering the MS the flow was split 1:3-   Gradient: The gradient was started at t=0 minutes with 100% (v/v) A,    remaining for 15 minutes and changed within 15 minutes to 80% (v/v)    B (t=30 minutes). From 30 to 31 minutes the gradient was kept at    constant at 80% (v/v) B.-   Injection volume: 5 μl-   MS detection: ESI(+)-MS    -   The electrospray ionization (ESI) was run in the positive scan        mode with the following conditions; m/z 50-500, 50 V fragmentor,        0.1 m/z step size, 350° C. drying gas temperature, 10 L N₂/min        drying gas, 50 psig nebuliser pressure and 2.5 kV capillary        voltage.

Cloning of Target Genes

Design of Expression Constructs

attB sites were added to all genes upstream of the ribosomal bindingsite and start codon and downstream of the stop codon to facilitatecloning using the Gateway technology (Invitrogen, Carlsbad, Calif.,USA).

Gene Synthesis and Construction of Plasmids

Synthetic genes were obtained from DNA2.0 and codon optimised forexpression in E. coli according to standard procedures of DNA2.0. Theaminotransferase genes from Vibrio fluvialis JS17 [SEQ ID No. 86] andBacillus weihenstephanensis KBAB4 [SEQ ID No. 89] encoding the aminoacid sequences of the V. fluvialis JS17 ω-aminotransferase [SEQ ID No.82] and the B. weihenstephanensis KBAB4 aminotransferase(ZP_(—)01186960) [SEQ ID No. 83], respectively, were codon optimised andthe resulting sequences [SEQ ID No. 88] and [SEQ ID No. 91] wereobtained by DNA synthesis.

Cells provided with said genes are referred to herein below as E. coliTOP10/pBAD-Vfl_AT and E. coli TOP10/pBAD-Bwe_AT respectively

Cloning by PCR

Various genes encoding a biocatalyst were amplified from genomic DNA byPCR using PCR Supermix High Fidelity (Invitrogen) according to themanufacturer's specifications

PCR reactions were analysed by agarose gel electrophoresis and PCRproducts of the correct size were eluted from the gel using the QIAquickPCR purification kit (Qiagen, Hilden, Germany). Purified PCR productswere cloned into pBAD/Myc-His-DEST expression vectors using the Gatewaytechnology (Invitrogen) via the introduced attB sites and pDONR-zeo(Invitrogen) as entry vector as described in the manufacturer'sprotocols. The sequence of genes cloned by PCR was verified by DNAsequencing. This way the expression vectors, pBAD-Bsu_gi16077991_AT(comprising a gene as represented by Sequence ID 133, encoding peptiderepresented by Sequence ID 134), pBAD-Pae_gi9946143_AT (using primers asidentified in sequence ID's 130 and 131), pBAD-Pae_gi9951072_AT(comprising a gene as represented by Sequence ID 135, encoding peptiderepresented by Sequence ID 136), pBAD-Pae_gi9951630_AT (comprising agene as represented by Sequence ID 137, encoding peptide represented bySequence ID 138) were obtained. The corresponding expression strainswere obtained by transformation of chemically competent E. coli TOP10(Invitrogen) with the pBAD constructs.

Enzymatic Reactions for Conversion of 5-Formylpentanoic Acid to 6-ACA

Unless specified otherwise, a reaction mixture was prepared comprising10 mM 5-formylpentanoic acid, 20 mM racemic α-methylbenzylamine, and 200μM pyridoxal 5′-phosphate in 50 mM potassium phosphate buffer, pH 7.0.100 μl of the reaction mixture were dispensed into each well of the wellplates. To start the reaction, 20 μl of the cell free extracts wereadded, to each of the wells. Reaction mixtures were incubated on ashaker at 37° C. for 24 h. Furthermore, a chemical blank mixture(without cell free extract) and a biological blank (E. coli TOP10 withpBAD/Myc-His C) were incubated under the same conditions. Samples wereanalysed by HPLC-MS. The results are summarised in the following table.

TABLE 3 6-ACA formation from 5-FVA in the presence of aminotransferases6-ACA Biocatalyst concentration [mg/kg] E. coli TOP10/pBAD-Vfl_AT 43* E.coli TOP10/pBAD-Pae_pBAD- 930  Pae_gi_9946143 E. coli TOP10/pBAD-Pae_AT25* E. coli TOP10/pBAD-Bwe_AT 24* E. coli TOP10/pBAD-Bsu_gi16077991_AT288  E. coli TOP10/pBAD-Pae_gi9951072_AT 1087   E. coliTOP10/pBAD-Pae_gi9951630_AT 92  E. coli TOP10 with pBAD/Myc-His C(biological   0.6 blank) None (chemical blank) not detectable *methoddiffered in that 10 μl cell free extract was used instead of 20 μl, thepyridoxal-5′-phosphate concentration was 50 μM instead of 200 μM and thereaction mixture volume in the wells was 190 μl instead of 100 μl

It is shown that 6-ACA is formed from 5-FVA in the presence of anaminotransferase, and that E. coli is capable of catalysing thisformation.

1. Method for preparing an adipate ester or adipate thioester,comprising converting a 2,3-dehydroadipate ester or 2,3-dehydroadipatethioester into the adipate ester or thioester in the presence of abiocatalyst.
 2. Method according to claim 1, wherein the biocatalystcomprises an enzyme capable of catalysing the reduction of acarbon-carbon double bond of a 2,3-enoate moiety or a 2-enoyl moiety. 3.Method according to claim 2, wherein the biocatalyst comprises an enzymeselected from the group of oxidoreductases acting on the HC—CH group ofdonors (EC 1.3.1. or 1.3.99), preferably from the group ofoxidoreductases (EC 1.3.1 and EC 1.3.99), preferably from the group ofenoyl-CoA reductases EC 1.3.1.8, EC 1.3.1.38 and EC 1.3.1.44, from thegroup of enoyl-[acyl-carrier-protein] reductases EC 1.3.1.9, EC 1.3.1.10and EC 1.3.1.39, and from the group butyryl-CoA dehydrogenase (EC1.3.99.2), acyl-CoA dehydrogenase (1.3.99.3) and long-chain-acyl-CoAdehydrogenase (EC 1.3.99.13).
 4. Method according to claim 1, whereinthe biocatalyst comprises an enzyme, which enzyme comprises an aminoacid sequence selected from the group of amino acid sequencesrepresented by any of the SEQUENCE ID's 42-67, 94, 96, 98, 100, 102,103, 105, 106, 107, 109, 111, 113, 115, 116 and homologues thereof, inparticular an amino acid sequence selected from the group of amino acidsequences represented by any of the SEQUENCE ID's 60, 63, 96, 100 andhomologues thereof.
 5. Method according to claim 1, wherein thebiocatalyst comprises an enzyme of an organism selected from the groupof Escherichia (in particular E. coli), Vibrio, Bacillus (in particularB. subtilis), Clostridia (in particular C. kluyveri, C. acetobutylicumand C. perfringens), Streptomyces (in particular S. coelicolor and S.avermitilis), Pseudomonas (in particular P. putida and P. aeruginosa),Shewanella, Xanthomonas, Xylella, Yersinia, Treponema (in particular T.denticola), Eubacterium (in particular E. pyruvativorans), Micorscilla(in particular Micorscilla marina), Aeromonas (in particular Aeromonashydrophila), Megasphera (in particular Megasphera elsdenii),Acinetobacter sp., Deinococcus (in particular Deinococcus radiourans),Yarrowia (in particular Yarrowia lypolytica), Euglenozoa (in particularEuglena gracilis), Saccharomyces (in particular S. cerevisiae),Kluyveromyces (in particular K. lactis), Schizosaccharomyces (inparticular S. pombe), Candida (in particular C. tropicalis), Aspergillus(in particular A. niger and A. nidulans), Penicillium (in particular P.chrysogenum), Arabidopsis (in particular A. thaliana), Homo sapiens,Rattus norvegicus, Bos Taurus, Cavia sp., Caenorhabditis elegans, andDrosophila melanogaster.
 6. Method according to claim 1, wherein2,3-dehydroadipate ester or 2,3-dehydroadipate thioester is prepared byconverting a 3-hydroxyadipate ester or 3-hydroxyadipate thioester. 7.Method according to claim 6, wherein the 3-hydroxyadipate ester orthioester is biocatalytically converted in the presence of a biocatalystcapable of catalysing the dehydration of a 3-hydroxyacyl ester or3-hydroxyacyl thioester to a 2-enoyl ester or thioester, preferably abiocatalyst comprising an enzyme selected from the group of hydrolyases(EC. 4.2.1), preferably from the group of enoyl-CoA hydratases (EC4.2.1.17), 3-hydroxybutyryl-CoA dehydratases (EC 4.2.1.55) andlong-chain-enoyl-CoA hydratases (EC 4.2.1.74).
 8. Method according toclaim 7, wherein said biocatalyst comprises an enzyme of an organismselected from the group of Acinetobacter (in particular Acinetobactersp. strain ADP1 and A. calcoaceticus), Alicaligenes (in particularAlicaligenes D2), Aspergillus (in particular A. niger), Azoarcus (inparticular A. evansii), Bacillus (in particular B. halodurans),Corynebacterium (in particular C. glutamicum and C. aurantiacum), E.coli, Flavobacterium, Neurospora (in particular N. crassa), Penicillium(in particular P. chrysogenum), Pseudomonas (in particular P. putida andP. fluorescens), Rhodopseudomonas (in particular R. palustris),Rhodococcus (in particular Rhodococcus sp strain RHA1), Aeromonas (inparticular A. caviae), Clostridium (in particular C. acetobutylicumi andC. kluyveri), Gossypium (in particular G. hirsutum), Rhodospirillum (inparticular R. rubrumi), and Ralstonia (in particular Ralstoniaeutropha), Euglenozoa (in particular Euglena gracilis, Megasphera (inparticular M. elsdenii), and Saccharomyces (in particular S.cerevisiae), and mammals (in particular Bos taurus, Homo sapiens, Rattusnorvegicus, and Sus scrofa).
 9. Method according to claim 6, wherein the3-hydroxyadipate ester or thioester is prepared by converting a3-oxoadipate ester or 3-oxoadipate thioester.
 10. Method according toclaim 9, comprising biocatalytically converting the 3-oxoadipate esteror 3-oxoadipate thioester, in the presence of a biocatalyst, inparticular a biocatalyst capable of catalysing the reduction of acarbonyl group to an alcohol group or capable of catalysing thereduction of a 3-oxoacyl ester or 3-oxoacyl thioester to thecorresponding 3-hydroxyacyl ester or thioester, selected from the groupof dehydrogenases (E.C. 1.1.1), preferably from the group3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35 and EC 1.1.1.36),3-hydroxybutanoyl-CoA dehydrogenase (EC 1.1.1.157),3-hydroxypimeloyl-CoA dehydrogenase (EC 1.1.1.259) andlong-chain-3-hydroxyacyl-CoA dehydrogenases (EC 1.1.1.211).
 11. Methodaccording to claim 10, wherein said biocatalyst comprises an enzyme ofan organism selected from the group of Acinetobacter (in particularAcinetobacter sp. Strain ADP1 and A. calcoaceticus), Alicaligenes (inparticular Alicaligenes strain D2 and A. eutrophus), Arzoarcus (inparticular A. evansii), Bacillus (in particular B. halodurans),Bordetella (in particular B. pertussis), Burkholderia (in particular B.pseudomallei and B. xenovorans), Corynebacterium (in particular C.glutamicum, C. aurantiacum and C. efficiens), Deinococcus (in particularD. radiodurans), E. coli, Flavobacterium, Klebsiella (in particular K.pneumonia), Pseudomonas (in particular P. putida and P. fluorescens),Rhodopseudomonas (in particular R. palustris), Rodococcus (in particularR. erythropolis, R. opacus, and Rodococcus sp strain RHA1), Aspergillus(in particular A. niger), Neurospora (in particular N. crassa),Penicillium (in particular P. chrysogenum), Saccharomyces, Bos taurus,Rattus norvegicus, Sus scrofa, Homo sapiens. Clostridia (in particularC. acetobutylicum and C. kluyveri), Euglenozoa (in particular) Euglenagracilis, Megasphera (in particular Megasphera elsdenii), Ralstonia (inparticular Ralstonia eutropha), and Zoogloea (in particular Zoogloearamigera).
 12. Method according to claim 9, wherein the 3-oxoadipateester or 3-oxoadipate thioester is prepared by reacting a succinateester or succinate thioester with an acetate ester or acetate thioester.13. Method according to claim 12, comprising biocatalytically reacting asuccinate ester or thioester with a acetate ester or thioester, in thepresence of a biocatalyst, preferably a biocatalyst comprising an enzymecapable of acetyl-group transfer selected from the group ofacyltransferases (E.C. 2.3.1), in particular an acyltransferase from thegroup of acetyl-CoA:acetyl-CoA C-acetyltransferases (EC 23.1.9),acyl-CoA:acetyl-CoA C-acyltransferases (EC 2.3.1.16) andsuccinyl-CoA:acetyl-CoA C-succinyltransferases (EC 2.3.1.174), more inparticular an enzyme comprising an amino acid sequence as identified inany of the SEQUENCE ID's 1-13, or a homologue thereof.
 14. Methodaccording to claim 12, wherein said biocatalyst comprises an enzyme ofan organism selected from the group of, Acinetobacter (in particularAcinetobacter sp. Strain ADP1 and A. calcoaceticus), Agrobacterium (inparticular A. tumefaciens), Alicaligenes (in particular Alicaligenesstrains D2 and A. eutrophus), Arthrobacter, Arzoarcus (in particular A.evansii), Azomonas, Azotobacter, Bacillus (in particular B. halodurans),Beijerinckia, Bradyrhizobium, Burkholderia, Clostridia (in particular C.kluyveri), Comamonas, Corynebacterium (in particular C. glutamicum andC. aurantiacum), E. coli, Enterobacter, Flavobacterium, Megasphera (inparticular M. elsdenii), Norcadia, Pseudomonas (in particular P. putida,P. aeruginosa and Pseudomonas sp. strain B13), Ralstonia (in particularR. eutropha), Rhizobium, Rhodopseudomonas (in particular R. palustris),Rodococcus (in particular R. erythropolis, R. opacus, and Rodococcus spstrain RHA1), Aspergillus (in particular A. niger), Euglenozoa (inparticular Euglena gracilis), Neurospora (in particular N. crassa),Penicillium (in particular P. chrysogenum), Rhodotorula, Saccharomyces,Trichosporon (in particular T. cutaneum).
 15. Method according to claim1, wherein any of said esters is selected from the group of biologicalactivating groups, in particular from the group of coenzyme A,phospho-pantetheine, which may be bound to an acyl or peptidyl carrierprotein, N-acetyl-cysteamine, methyl-thio-glycolate,methyl-mercapto-propionate, ethyl-mercapto-propionate,methyl-mercapto-butyrate, methyl-mercapto-butyrate andmercaptopropionate.
 16. Method for preparing adipic acid comprisingpreparing adipate ester or adipate thioester in a method according toclaim 1, and hydrolysing the adipate ester or adipate thioester obtainedin a method according to any of the preceding claims, wherein thehydrolysis is preferably catalysed by a biocatalyst, in particular by abiocatalyst comprising an enzyme selected from the group of hydrolases(EC 3.1.2).
 17. Method for preparing adipic acid, comprising preparingadipate ester or adipate thioester in a method according to claim 1, andtransferring the activating group of the adipate ester or adipatethioester obtained wherein the activating group transfer is catalysed bya biocatalyst, preferably a biocatalyst comprising an enzyme catalysingthe transfer of sulphur-containing groups (EC 2.8), more preferably fromthe group of CoA transferases (EC 2.8.3).
 18. Method for preparing5-formylpentanoate comprising preparing adipate ester or adipatethioester in a method according to claim 1, or preparing adipic acid andconverting the adipate ester, the adipate thioester or the adipic acidinto 5-formylpentanoate.
 19. Method according to claim 18, wherein theconversion into 5-formylpentanoate is catalysed by a biocatalyst,preferably a biocatalyst comprising an enzyme selected from the group ofoxidoreductases (EC 1.2.1), preferably from the group of aldehydedehydrogenase (EC 1.2.1.3, EC 1.2.1.4 and EC malonate-semialdehydedehydrogenase (EC 1.2.1.15), succinate-semialdehyde dehydrogenase (EC1.2.1.16 and EC 1.2.1.24); glutarate-semialdehyde dehydrogenase (EC1.2.1.20), aminoadipate semialdehyde dehydrogenase (EC 1.2.1.31),adipate semialdehyde dehydrogenase (EC 1.2.1.63) or from the group ofaldehyde dehydrogenases (acetylating) (EC 1.2.1.10), fatty acyl-CoAreductases (EC 1.2.1.42), long-chain-fatty-acyl-CoA reductases (EC1.2.1.50), butanal dehydrogenases (EC 1.2.1.57) and succinatesemialdehyde dehydrogenases (acetylating).
 20. Method for preparing6-amino caproic acid, comprising preparing 5-formylpentanoate in amethod according to claim 18, and converting the 5-formylpentanoate into6-amino caproic acid.
 21. Method according to claim 20, wherein theconversion is catalysed by a biocatalyst, in particular a biocatalystcapable of catalysing a transamination and/or a reductive amination,preferably a biocatalyst comprising an enzyme selected from the group ofaminotransferases (EC 2.6.1) and amino acid dehydrogenases (EC 1.4.1),more preferably from the group of β-aminoisobutyrate:α-ketoglutarateaminotransferases, β-alanine aminotransferases, aspartateaminotransferases, 4-amino-butyrate aminotransferases (EC 2.6.1.19),L-lysine 6-aminotransferase (EC 2.6.1.36), 2-aminoadipateaminotransferases (EC 2.6.1.39), 5-aminovalerate aminotransferases (EC2.6.1.48), 2-aminohexanoate aminotransferases (EC 2.6.1.67),lysine:pyruvate 6-aminotransferases (EC 2.6.1.71), andlysine-6-dehydrogenases (EC 1.4.1.18).
 22. Method according to claim 20,wherein an aminotransferase is used comprising an amino acid sequenceaccording to Sequence ID 82, Sequence ID 83, Sequence ID 84, Sequence ID134, Sequence ID 136, Sequence ID 138 or a homologue of any of thesesequences.
 23. Method according to claim 20, wherein the biocatalystcomprises an enzyme from an organism selected from the group of Vibrio;Pseudomonas; Bacillus; Mercurialis; Asplenium; Ceratonia; mammals;Neurospora; Escherichia; Thermus; Saccharomyces; Brevibacterium;Corynebacterium; Proteus; Agrobacterium; Geobacillus; Acinetobacter;Ralstonia and Salmonella.
 24. Method for preparing caprolactam,comprising preparing 6-amino caproic acid in a method according to claim20, and cyclising the 6-amino caproic acid, thereby forming caprolactam.25. A host cell comprising one or more nucleic acid sequences encodingan enzyme as defined in claim 2, preferably comprising at least twonucleic acid sequences each encoding a different enzyme.
 26. A host cellaccording to claim 25, comprising a nucleic acid sequence encoding apolypeptide as represented by any of the Sequence ID's 42-67, SequenceID's 74-81, 94, 96, 98, 100, 102, 103, 105, 107, 109, 111, 113, 115, 116or a homologue thereof.
 27. Method according to claim 1, comprisingcontacting cells comprising the biocatalyst—which biocatalyst maycomprise a host cell or a different biocatalyst—with a fermentablecarbon source, wherein the carbon source contains any of said compoundswhich are to be converted into the compound to be prepared or whereinthe cells prepare the compound to be converted into the compound to beprepared from the carbon source.
 28. Method for preparing adipic acidfrom succinic acid or an ester or thioester of succinic acid thereof,optionally according to claim 16, via a plurality of reactions, whereinat least one of the reactions is catalysed by a biocatalyst.
 29. Methodaccording to claim 28, comprising (1) providing a succinate ester orthioester and reacting said ester or thioester with an acetate ester orthioester, thereby forming a 3-oxoadipate ester or thioester; (2)hydrogenating the 3-oxo group of the 3-oxoadipate ester or thioesterthereby forming a 3-hydroxyadipate ester or thioester; (3) dehydratingthe 3-hydroxyadipate ester or thioester thereby forming a 2,3-dehydroadipate ester or thioester; (4) hydrogenating of the C—C double bond ofthe 2,3-dehydro adipate ester or thioester, thereby forming an adipateester or thioester; and (5) hydrolysing the ester bond or thioesterbond, thereby forming adipic acid.
 30. Polypeptide comprising an aminoacid sequence according to any of the Sequence ID's 57, 68, 69, 70, 71,72, 79, 85, and homologues thereof.
 31. Polynucleotide, encoding apolypeptide according to claim 30.